The Normal Human Liver Cell - NCBI

The Normal Human Liver Cell

Cytochemical and Ultrastructural Studies

M. H. Ma, MB, BS and L. Biempica, MD

MORPHOLOGIC 1 AND HISTOCHEMICAL 1-3 HETEROGENEITY ofcells within the hepatic lobule of the rat is well known, as is thefrequently zonal nature of various hepatic injuries in animals of allspecies. It is therefore surprising that few of the many ultrastructuralstudies of the diseased human liver and none of those dealing with thenormal human liver 4-9 have been concerned with intralobular differ-ences. Further, there have been no combined ultrastructural and cyto-chemical studies of the normal human hepatocyte.

Cytochemical staining methods, with their precise localizations ofcertain enzymes in the various cell components, serve to integratebiochemical information with cellular morphology. Thus, electronmicroscopy and enzyme cytochemistry were simultaneously applied toa study of liver biopsies collected during the last 7 years from peoplewho were shown to be without hepatic dysfunction detectable by thetechnics currently available. Particular attention was directed at dif-ferences between cells from different lobular zones. This work wasaimed toward a better understanding of the range of "modulation" ofthe normal hepatocyte and to provide a baseline for a more meaningfulanalysis of the abnormal hepatocyte.

Materials and MethodsEight males and 4 females, ranging in age from 9 months to 53 years, were biop-

sied by their attending physicians. Seven of the biopsies were indicated in thecourse of exhaustive investigations for symptoms possibly of hepatic origin, but latershown to be unrelated to the liver. The indication for biopsies in the remainder wasfor excluding familial diseases, such as hemochromatosis or Wilson's disease, becauseone or another member of the families suffered from these diseases. All patientswere subsequently considered to be without hepatic dysfunction by the following

From the Departments of Pathology and Medicine, Albert Einstein College of Medi-cine, Bronx, New York.

Supported by Grants AM-10852 and CA-06576 from the US Public Health Service,the New South Wales State Cancer Council and Postgraduate Medical Foundation ofSydney University, Sydney, Australia.

Accepted for publication November 9, 1970.Address for reprint requests: Dr. Marcus H. Ma, Department of Pathology, Albert

Einstein College of Medicine of Yeshiva University, 1300 Morris Park Avenue, Bronx,New York 10461.

353

354 MA AND BIEMPICA American Journalof Pathology

criteria: (1) the absence of stigmata of liver disease oIn physical examination; (2)normal tests for serum proteins, bilirubin. alkaline phosphatase. glutamic oxalacetictransaminase, glutamic pymtvic transaminase and bromsulphthalein retention at 45minutes; (3) normal findings in histologic exaniiiiatioii of paraffini-embedded for-malin-fixed liver tissues. Biopsies w-ere done under local anesthesia using either aFrancine or eienghini needle.

Cytochemical Studies

Tissues wvere fixed overnight at 0-4 C in 4A formaldehyde-1¶ CaCl "' or for1.5-3 hours in 2.5 or 3% glutaraldehvde 11 in 0.1 NM cacodvlate at pH 7.4.

For light microscopy, 10-u-thick frozen sections were incubated at 37 C to dem-onstrate various organelle marker-enzymes, usinig media containing the folloxvingsubstrates:

1. For acid phosphatase, P-glucuronidase and glucosaminidase in lvsosomes:cvtosine-5'-monophosphate (CMP),12 naphthol AS-TR phosphate.'3 naphthol AS-BI3-D-glucuronide,' N-acetyl-p-n-glucosamine naphthol AS-LC,14 all at pH 5.2. For nucleoside phosphatases and alkalinie phosphatase in the plasma mem-

brane: adenosine triphosphate (ATP),15 adenosine monophosphate (ANIP) .1 cv~-tosine-5.'-monophosphate wvith cobalt 16 (cobalt-stimulated CMIP) at pH 7.2 andct-x3 glycerophosphate 17 at pH 9.4.

3. For nucleoside diphosphatase in endoplasmic reticulum: inosine diphosphate(IDP) .'I

4. For thiamine pyrophosphatase in the Golgi apparattus: thiamine pyrophosphate(TPP). 18

5. For reductases in mitochondria: P3-dihydronicotinamide adenine dinucleotidewith tetranitro blue tetrazolium (NADHI-TNBT) at pH 7.0 19 and for mitochondrialcvtochromes (probably) with 3',3'-diaminobenzidine (DAB) at pH 6.0.2'"

6. For catalase in peroxisomes (microbodies): DAB at pH 9.0.2I

For all reactions, both glutaraldehvde-fixed and formaldehyde-fixed tissues wereused, but glutaraldehbde was preferred because it preserved tissue better.

For electron microscopy. 25-L nonfrozen "chopper" sections1 wvere preparedfrom three biopsy samples for incubation in the DAB media for %isualizing peroxi-somes and mitochondria, and from one biopsy for incubation in CNIP. IDP an(dTPP media. Incubated tissues were postfixed for 1 hour at 4 C in Veronal-buffered1% OSO, -22 and processed as for morphologic studies.

Morphologic Ultrastructural Studies

Tissues -vere cut into approximately 1-mm pieces and immersed in the follomNingcold fixatives: (1) 1-2% OsO, in 0.1 MI Soren.seni's phosphate2 buffer for 2 hours,(2) 2_ OsO, in Veronal buffer -2 containiing 0.2 MI suicrose for 2 hours, or (3)2.5% glutaraldehvde in 0.1 MI cacodylate or 0.1 MI phosphate buffer for 2 hours '1then rinsed in the same buffer and postfixed for 1 hour in phosphate or veronal-buffered 1% OsO,. Tissues wvere dehvdrated at room temperatures in graded alco-hols and propylene oxide, and wvere embedded in either Araldite or Epon.24 Sometissues were also rinsed in uranyl acetate before dehydration and embedding.')One-micron sections, cut from the Epon blocks and stained with toluiidine bliue.were used to select appropriate areas for ultrathini sectioning. At least one peri-portal and one centrolobular area xvere located from each of 8 biopsies for electronmicroscopic examination. Unfortunatel,% portal tracts and central veins vvere not

Vol. 62, No. 3 STUDIES OF NORMAL HUMAN LIVER CELLS 355March 1971

included in the small amounts of material available from the remaining 4 biopsies,which thus had to be examined at random. For the purpose of this study periportalrefers to a small area surrounding a portal triad containing a vein, artery and bileduct. Since no attempt was made to differentiate between small or large portalspaces, our periportal areas may or may not correspond to the zone 1 of Rappa-port.26 Ultrathin sections were mounted on uncoated grids except for serial sections,which were picked up on formvar-supported single-hole grids. Sections were stainedwith lead citrate 27 or with uranyl acetate 28 anid then lead and examined in a Sie-mens Elmiskop I at 80 kV.

Results

Plasma Membrane

The intensity of staining of the plasma membranie for various phos-phatases varied. In addition, there were striking differences in stainingbetween the sinusoidal and bile canalicular membranes. Suchstaining was not demonstrable on the lateral intercellular portions ofthe plasma membrane. The sinusoidal aspect stained intensely fornucleoside phosphatase with AMP (Fig 1), CMP (Co++ stimulated)pH 7.2; and alkaline phosphatase with glycerophosphate, but little andsometimes no staining with ADP or ATP was observed (Fig 2). Onthe other hand, the bile canaliculi stained strongly with ATP (Fig 2),IDP and AMP (Fig 1) but not for alkaline phosphatase activity. Stain-ing of canaliculi was generally more intense and the canalicular lumenwider in the periportal hepatocytes, but there was no apparent zonaldifference in sinusoidal activity.The surface of the sinusoidal plasma membrane was provided with

a variable number of long, often tortuous and sometimes bifurcatingmicrovilli (Fig 3A and 4). Consistent with the lack of zonal differencesin sinusoidal surface phosphatase activities, there was no difference innumber, length or degree of complexity in microvilli from differentlobular zones.The lateral surfaces on either side of the bile canaliculus were

generally devoid of microvilli except where the plasma membraneswere modified to form the bile canaliculi. Short nonbranching micro-villi were present along these lateral surfaces where bile canaliculi werenot observed (Fig 3). Microvilli in this location have not been describedin normal livers. Desmosomes were present along the lateral surfaces.They were composed of straight and strictly parallel, localized areas ofplasma membrane from two neighboring cells, reinforced on either sideby a parallel layer of cytoplasmic condensation or cytoplasmic plaque25and bundles of fine fibrils converging towards this plaque (Fig 5).A distinct intercellular plate was not seen with any of the fixatives used


in this studv, but rather a slightly electron-dense homogeneous materialfilling the intercellular space. Occasionallv, a mitochondrion was adja-cent to the fine fibrils at each side of the desmosome, forming a mirror-image appearance. This mitochondrion-desmosome complex has beenobserved in several epithelial cells, and in human hepatocv-tes.'A variable number of microvilli projected into the bile canaliculus.

Correlated with the weaker staining for nucleoside phosphatase,canaliculi in the centrilobular zone (Fig 12) appeared to have fewermicrovilli and a narrower lumen than those in the periportal zone(Fig 13).The presence of tight junctions is well established in the livers of

animals30 and recentlv also in man.7 They are formed bv a fusion of theexternal leaflets of the neighboring plasma membranes at the lateralextremities of the bile canaliculi (Fig 6). Next to the tight junction,an intermediate junction was consistently present (Fig 6). Here a trueintercellular space was found and the opposing plasma membranestended to run a wavy course, often not parallel, in contrast to thatdescribed in other cell tvpes.'5 It characteristically possessed an adjacentzone of condensed and amorphous cvtoplasmic material, which oftenextended to become part of the tight junction. Immediatelv distal tothe intermediate junction, a desmosome was found only infrequently,in contrast to the junctional complexes of other cell types.A zone of specialized "ectoplasm," approximatelv 0.2 [t in thickness,

surrounded the bile canaliculi. This zone was composed of compact,finely divided material. It was characteristically lacking in identifiableorganelles except for a few minute vesicles and short tubular structures(Fig 6 and 7). These have been described by Biava, who considersthat thev are formed as a result of progressive dissolution of SER andGolgi vesicles in the process of bile secretion.7 In addition, larger single-membrane-limited vacuoles, approximately 100-150 m1j in diameter,were found in the pericanalicular zone of about one third of the bilecanaliculi (Fig 7). These vacuoles have not been described previouslv.Thev were roughlv spherical and electron lucent, but sometimes thevhad a peripheral rim of faintly electron-dense content. The majoritvof these vacuoles were located in the cv-toplasmic zone immediatelvoutside the pericanalicular ectoplasm, but some were also seen withinthe ectoplasm. Thev differed from the SER, which occurred as eithershort, branching tubules or as irregularlv shaped vesicles filled with ahomogeneous, slightlv electron-dense material. No connection betweenthese vacuoles and the canalicular lumen wvas seen even in serialsections.


Nucleus

Since the fine structure of the human hepatocyte nucleus does notdiffer significantly from that of the rat liver 31 or other cells, it will beillustrated (Fig 8 and 9) rather than described in detail.

In a few periportal cells of one biopsy, large intranuclear glycogendeposits were found (Fig 10). This glycogen was of the monopartic-ulate type, occurring as single particles withouit stubtunits. Examinationof serial sections disclosed that they were wholly within the nuclei,without a limiting membrane or connection with the cytoplasm. Aroulndsmaller glycogen deposits, a faintly fibrillar ring of unidentified naturewas sometimes observed (Fig 10).

Rough Endoplasmic Reticulum (RER)

In 4 cases, nucleoside diphosphatase activity was studied, with IDPas substrate, by light microscopy. It was difficult to identify the ERby light microscopy, even after histochemical staining. Small, brownish,threadlike areas probably represented the ER (Fig llA), but they werenot readily visualized and only rarely were clumps similar to those ofrats seen. Lobular differences were not evident.The nucleoside diphosphatase reaction was examined by electron

microscopy in 1 case (Fig I1B). Electron-dense amorphous reactionproduct was demonstrated inside most, but not all ER cisternae.Reaction product was also present in the nuclear envelope and in thefirst one or two saccules on the "concave" aspect of the Golgi stack.By electron microscopy of unincubated tissues, parallel arrays of

RER cisternae were not commonly fouind in the human liver cell. Whenthey occurred, the cisternae making up the arrays were seldom numerous.There was more RER in periportal (Fig 3 and 13) than centrolobularcells (Fig 12 and 22). The abundance of RER was especially evidentin the first two or three layers of cells immediately adjacent to theportal space. In the rat liver, the RER 32 and clumps of nucleosidediphosphatase reaction products representing parallel arrays of RER 33

are more abundant in centrolobular hepatocytes.The major portion of the RER appeared as single, or small groups

of, flattened sacs (Fig 12 and 13) in the vicinity of the mitochondria.WVherever mitochondria were found, the adjacent RER profiles wereoriented parallel to the surface of the mitochondria. In cells near thecentral vein (Fig 12) where RER was less plentiful, this close spatialrelationship between the RER and mitochondria was more readilyappreciated.The endoplasmic reticulum along the sinusoidal border was pre-


dominantly of the rough varietv (Fig 12, 13, and 21), in contrast tothat along the lateral cell borders, where variable amounts of bothSER and RER were present. The orientation of the RER profiles wvithrespect to the cell surface appeared to be random.The RER was frequentlv concentrated around the nucleus (Fig 12).

Here, the cisternae profiles tended to be oriented parallel to the cir-cular outline of the nucleus. The relationship of the RER to the Golgiapparatus will be discussed in a later section.The widths of the ER cisternae in all 7 biopsies fixed in 2r OsO in

either phosphate or Veronal buffer measured 250-40 A. In the biopsiesfixed in either 1% Os04 or 3ci glutaraldehvde, the widths of thecisternae varied from cell to cell and were often >400 A.

Smooth Endoplasmic Reticulum (SER) and Glycogen

In Os04-fixed tissues, the SER appeared most commonly as irregularlyshaped vesicular structures interposed among glvcogen rosettes (Fig12, 13 and 21). In some cells, or even in other parts of the same cell,the SER appeared as short, branching tubules. Occasionally, complexbranching or reticular formations were observed. The lumen of the SERwas moderately electron dense, but contained no particulate material.In glutaraldehyde-fixed tissues, the SER sometimes appeared moder-atelv dilated, and had a rounded contour and invaginations which, incross section, appeared as vesicles (Fig 13B).

Ribosomes were often observed to be attached to one portion of theendoplasmic reticulum and absent in a large part of another portionof the same cistema. These portions devoid of ribosomes w%ere inter-preted as sites of continuitv between RER and SER (Fig 21). Incontrast to the RER, the SER appeared more abundant in the centro-lobular (Fig 12) than in the periportal cells (Fig 3 and 13). Glycogenareas devoid of SER were rare as the two components were inter-mingled intimately.

Mitochondna

All hepatocytes displayed NADH,-TNBT reductase activity (Fig14). The level of activitv was distinctlv higher in the periportal zones.Mitochondria in the periportal zone (Fig 3 and 13) were morenumerous, and were often oval or oblong. 'Measurements from randommicrographs showed them to vary from 0.4 M in width by 0.7 [t in lengthto 0.6 [i by 1.0 [t. Mitochondria in centrolobular cells (Fig 12) tendedto be round or oval and less numerous than in periportal cells. Thevmeasured from 0.5 [i by 0.6 [t to 0.6 [t by 0.8 It.


In comparison with mitochondria of the rat liver,3' human mitochon-dria appeared to contain fewer cristae; these cristae did not appear tobe as distinctly oriented at right angles to the outer membrane. Mostmitochondria contained varying numbers of dense granules composedof tightly packed laminae of highly electron-dense material.34 The closespatial relationship of the mitochondria to the RER has been noted.Indeed, it was unusual to find mitochondria without accompanyingRER. Like the RER, therefore, the mitochondria were more concentratednear the nucleus and along the sinusoidal surface.

In 3 biopsies, two types of unusual mitochondria were found, albeitwith rarity. One type contained crystalline inclusions. The inclusionswere found in the larger mitochondria of periportal cells in 2 biopsies(Fig 17 and 18); they did not oxidize DAB (Fig 18). Curling of thecristae was observed in the other unusual mitochondria (seen in onecell of 1 biopsy) (Fig 19).

Incubation in the DAB medium at pH 6 revealed the presence ofoxidation reaction product within the mitochondrial cristae in all 3biopsies (Fig 15). Reaction product was also present in the outermitochondrial membrane in 1 of the biopsies but absent in the other2. The reaction has been attributed to cytochromes c and b.2035

Peroxisomes (Microbodies)36

Reaction product was found in the matrix of all peroxisomes seenin the 3 biopsies studied with DAB at pH 9.0 (Fig 20). With theexception of some mitochondria, no other structures were stained.Available evidence indicates that the peroxisomal oxidation of DAB isdue to catalase20 or its peroxidase subunits formed as a result of alkalinehydrolysis of catalase.'7Human hepatic peroxisomes are relatively round organelles contain-

ing a homogeneous, moderately electron-dense matrix limited by asingle membrane. Cores were found in several cells of 1 biopsy (Fig12 and 21A). The cores were more electron dense than the matrix andwere not crystalline but finely granular. Marginal plates seen in per-oxisomes of many species including those of cebus monkey hepatocytesand human renal tubular cells, were not seen in this study nor havethey been reported in human hepatocytes (see Hruban and RechCigl 38 ).

Characteristically, SER and glycogen were consistently located inclose proximity to the peroxisomes. Occasionally, peroxisomes possessedlong "tails," the membrane of which resembled that of the SER andwas continuous with it, as if forming from the latter 13 (Fig 21B).

Peroxisomes were 0.4-0.8 [t in diameter and were without apparent


intralobular difference in size. Almost invariably, mitochondria out-numbered peroxisomes by at least three to one. In approximatelv onefifth of cells in the first tw-o centrolobular layers of 1 biopsy, peroxisomesoutntumbered mitochondria (Fig 22), and in another 2 biopsies, perox-isomes %vere nearly as numerous as mitochondria in the same tw-olayers of centrolobular cells.

Golgi Apparatus

In frozen sections incubated for thiamine pvrophosphatase activity,the Golgi apparatus appeared as curved, sometimes hairpin-shapedthreads in the region of pericanalicular cvtoplasm (Fig 23).

In electron micrographs, sections of the Golgi apparatus showedstacks of parallel, generally cur-ed saccules (Fig 24-26). The Golgiapparatus thus had a convex and a concave surface. There were usually3 to 4 saccules, separated from each other by a relatively constantnarrow space. The saccules were usually dilated at their lateral ex-tremities btut may also show- dilatation elsew-here along their length.The saccules contained moderately electron-dense, irregularly shapedparticles, which w%ere more numerous w-ithin the dilated portions. Theseparticles have been most extensively- sttudied in rat li-er,39 where they-have been show-n to be very low density lipoproteins (VLDL).Many vesicles wvere seen in the reaion of the Golgi apparatuis. They

wvere found along both faces of the Golgi apparatus although thevwere more numerous along the convex surface. Some, particularly thosealong the convex surface of the Golgi stack, appeared to be movingfrom the adjacent RER to the Golgi saccules. 'Membrane continuitiesbetween x-esicles and RER and betw\een vesicles and the outer Golgisaccules wvere often encouintered (Fig 24). These vesicles wvere ap-proximately 500 A in diameter.

Larger vesicles, or ''vactuoles," of O.25-0.3 tt wvere less numerous thanthe smaller -esicles. They contained V'LDL particles and were usuallylocated near the concave surface of the Golgi apparatus (Fig 24-26).Some -acuoles were "coated" (Fig 26).

Adjacent to the concave surface of the Golgi apparatus, a specialsystem of smooth endoplasmic reticulum called GERL by Novikoff,44was identified (Fig 24 and 26). Its interconnecting cistemnae did notshow% the parallel arrangement that characterizes the Golgi sacctiles.Numerous V'LDL particles were present in GERL, particularly %vitbinits dilated regions. An outer coating -vas sometimes present along partsof these dilated regions (Fig 24). Apart from large VLDL-containingvacuoles, smaller ( 150-250 mit ) coated vesicles wvere also found in the


region of GERL. Acid phosphatase activity was demonstrable in GERLand in some nearby coated vesicles (Fig 36).The Golgi apparatuses of periportal cells differ from those of centro-

lobular cells in that the saccular dilatations from the periportal cellswere generally larger, more numerous and more often distended withVLDL (Fig 13A).

In the electron microscope, reaction products of thiamine pyrophos-phatase (Fig 27) and nucleoside diphosphatase (Fig 28) were local-ized to the first one or two, or rarely three, saccules on the concaveface. The reactions were more intense in the first saccules.

Lysosomes

Hepatocyte and Kupffer cell lysosomes were readily demonstrableby light microscopy, with either CMP (Fig 29 and 30) or 1-glucuronideas substrates. Hepatocyte lysosomes were smaller than Kupffer celllysosomes; they were seen predominantly near the bile canaliculi. Acidphosphatase and 0-glucuronidase activities were more intense in theperiportal than centrolobular hepatocytes and the lysosomes weregenerally larger there. Hepatocyte lysosomes did not stain for gluco-saminidase whereas those of some Kupffer cells did (Fig 31). Hayashi45has described weak but definite glucosaminidase activity in lysosomesof rat liver. This weak activity, compared with acid phosphataseactivity, was attributed to several factors including enzyme inhibitionby the diazo reagents used and the necessity to use low substrateconcentration because of poor substrate solubility.46 By electron micros-copy, several different forms of lysosomes were recognized: lipofuscingranules, autophagic vacuoles, hemosiderin granules, multivesicularbodies and acid phosphatase-containing vesicles in the Golgi region.

Lipofuscin granules are the most commonly encountered lysosomesin human hepatocytes. The ultrastructure of these granules has beendescribed.7'9'47 In their most typical form, they were single-membrane-limited bodies containing clumps of extremely dense material, varyingnumbers of relatively electron-lucid globules and an amorphous, moder-ately electron-dense matrix (Fig 3, 12, 13, and 32). The relative pro-portions of the three components varied widely.

All biopsies revealed autophagic vacuoles but only after a deliberatesearch for them (Fig 12 and 32). Various organelles have been identi-fied within these vacuoles, including mitochondria (most commonly),endoplasmic reticulum, small unidentified vesicles, glycogen, and ferri-tin. Membranous myelin-like material and electron-dense debris in-distinguishable from materials in lipofuscin granules were also seen


wvithin autophagic vacuoles. Autophagic vacuoles containing easilvrecognizable organelles usually had a double, smooth delimiting mem-brane (AV, in Fig 32), but vacuoles in a more advanced stage ofdegradation had onlv a single delimiting membrane (AN.. in Fig 32),(Novikoff and Shin 33)Hemosiderin granules are composed of closely packed ferritin par-

ticles bound by a single membrane (Fig 33). They were found in asmall number of cells in 2 biopsies, both from men over 350. In thesame livers, sparse but widely scattered ferritin particles were alsoseen free in the cytoplasm. These particles are considered to be ferritinon purely morphologic grounds.Y Some hemosiderin granules also con-tained partlv digested mitochondria, ER and other electron-opaquematerials similar to those seen in autophagic vacuoles.

Multivesicular bodies (M1VB) are membrane-delimited bodies con-taining varying numbers of small vesicles and a small amount of floc-culent, slightly electron-opaque material (Fig 3.3 and .34). Usuallytwo or three w%vere seen in each thin section of a cell (Fig 25). In theimmediate vicinity of this type of NMVB, two forms of structures werepresent: (1) small vesicles similar to some of those within the MIVB;(2) smooth tubular structures containing a slightly electron-opaquematerial. Frequently, projections from the surface of the NMVB in theform of rounded buds or tails were seen (Fig .34). 'Multivesicular bodiesapparently may function in autophagv and heterophag-, but the originof the vesicles they contain49 5t' is uncertain.Acid phosphatase reaction products w-ere found in this studx in all

of the following types of lvsosomes: lipofuscin granules (Fig 37), auto-phagic vacuoles (Fig 36) and multivesicular bodies (Fig 37). Thebiopsies 'with hemosiderin-containing cells w-ere not incubated for acidphosphatase activity.

Discussion

In this discussion, possible structure-function relations in humanhepatocvTtes and those described for rats and other animals will beemphasized.

Interchange Between Hepatocyte and Blood

Because of the multiplicity of hepatic functionis, extensive molecularinterchange must take place across the sinusoidal surfaces and perhapsalso across the lateral surfaces. The microvilli undoubtedly provide anincreased surface area for such an interchange.

Nucleoside phosphatases in plasma membranes occur wvidely in ani-


mal cells; high levels are found in specialized areas of the plasma mem-brane such as microvilli.15 It has been suggested that these phosphatasesparticipate in active transport but, so far, no direct evidence for thishas been obtained. Similarly, the proposed role of surface alkalinephosphatase in molecular transport has not been established (for areview see Ref 15). High levels of alkaline phosphatase activity arefound in only a relatively few cell types such as the brush border ofthe intestinal columnar cells and renal tubular cells and on theluminal surface of some capillaries. The mainly absorptive nature ofthese surfaces suggests that alkaline phosphatase may be more closelyassociated with absorption, rather than secretion. The intense stainingfor alkaline phosphatase of the hepatic sinusoidal surface (absorptive aswell as secretory) and the lack of staining of the canalicular sturface(mainly secretory) observed in this study and in previous studies in manand rabbits51 52 are consistent with this speculation. However, physiologicinterpretations about humans, such as these, are difficult to substantiateexperimentally since, in rats and guinea pigs, little or no sinusoidalsurface alkaline phosphatase is detectable by staining methods.51'52

Coated vesicles seen beneath the sinusoidal surface of the hepatocyteare similar to those seen in many other cell types. They are generallyconsidered to be pinocytic vesicles involved in the selective uptake ofproteins,5-1 but their role in the hepatocyte has not been established.

Success in the morphologic identification of very low density lipo-protein (VLDL) particles in the rat liver has permitted study of themode of secretion of these particles. The evidence indicates that VLDLis delivered to the sinusoidal surface in relatively large vacuoles, whichthen fuse with the sinusoidal membrane and discharge their contentsinto the space of Disse.39'42 In our material, VLDL-containing vacuolesare rarely seen toward the sinusoidal pole presumably because of therelatively slow rate of secretion of VLDL in the normal man.

It is possible that material synthesized in the ER may be secretedinto the space of Disse directly without being "packaged" into vacuoles.Thus, the accumulation of ER, particularly RER, beneath the sinu-soidal surface that was observed in this study may be an expression ofsuch a route of transport.

Bile Secretion

Recently, two cytoplasmic soluble-protein fractions, called Y and Z,have been shown to bind bilirubin as well as Bromsulphthalein.54These fractions may play an important role in the uptake of bilirubin.54The bilirubin-conjugating enzyme is present in the smooth endoplasmic


reticulum.55 A general correlation was noted between the presence ofnucleoside diphosphatase activity in the endoplasmic reticulum of rattissues and biochemicallv demonstrable glucuronide transferase activ-itv, which suggests some fuinctional relationship for these two enzvmeactivities.15The problem of organelle carriers for bilirubin and other bile con-

stituents is unsettled. Results of cell fractionation studies of the liversof rats injected with tritium-labeled bilirubin do not support the conceptof organelle carriers for bilirubin.J57

Studies of patients with the Dubin-Johnson syndrome and of mutantCorriedale sheep with a closely similar disorder of organic anionexcretion have suggested that hepatic organic anion excretion mavinvolve multiple excretorv mechanisms.5' As in the case of bilirubin,whether organelle carriers are involved in the transport of other organicanions to the bile canaliculus is not known.

In this study, a group of relativelv large vacuoles (100-150 m1t) inthe vicinity of the bile canaliculus wvere observed. Their close proximity%to the bile canaliculus suggests that they mav be involved in the secre-tion of some bile constituents. Our unpublished observations on 5biopsies from patients with complete obstruction of the common bileduct, showing the marked reduction or absence of these pericanalicularvacuoles, are consistent with this speculation.

Transport Within the Hepatocyte

This section 'will deal mainlyr with the possible mechanisms of trans-port of materials from the ER to the Golgi apparatus, V'LDL-contain-ing vacuoles, lysosomes and peroxisomes. The structural relations of theER and mitochondria, whose functional significance is much less under-stood, will also be brieflv discussed.Along the convex surface of the Golgi stack, an accumulation of

small vesicles, some of which appear to be derived by budding fromthe adjacent RER, have been described. It is likelv that materials aretransported from the ER to the Golgi saccules via these vesicles, as inguinea pig pancreas, where they apparently go to condensing vacuolesin the Golgi zone.59 Transport via small vesicles from ER to Golgisaccules has been suggested for cells of the rat epididvmis,60 eosino-phils,61 and protein-secreting cells such as thvroid follicular cells.62The material transported in this manner probably includes the triglv-

ceride and apoprotein moieties of VLDL.In rat liver cells, there is evidence for the transport of triglyrceride

from ER to the Golgi apparatus, and the packaging of VLDL in the


Golgi saccules.3942 As the apoprotein moiety of VLDL is a protein, itpresumably is synthesized in the ER and then transported to the Golgiappartus. Since it has not been established whether the apoproteinand triglyceride moieties of the VLDL are already coupled beforereaching the Golgi apparatus and since carbohydrates are known to bepart of the VLDL molecule," the packaging of VLDL in the Golgiapparatus may involve the coupling of apoprotein to triglyceride and/orthe addition of carbohydrate to the VLDL molecule. The addition, inthe Golgi zone, of certain sugars to the proteins in the formation ofglycoproteins is well established.64 Our observations that GERL, aswell as the Golgi saccules, contain large numbers of VLDL particlessuggests that VLDL may be also packaged in GERL. Further, theclose proximity of VLDL-containing vacuoles to GERL, and the pres-ence of an outer coating on the membranes of both VLDL-containingvacuoles and certain regions of GERL, suggest that at least some ofthese vacuoles are derived by budding from GERL.

Lysosomal enzymes are presumably synthesized in the ER. Morpho-logic and cytochemical studies, chiefly by Novikoff and his colleagues,suggest that the hydrolases are transported via GERL to various typesof lysosomes, where the hydrolases are concentrated.44 This concept issupported by our observation, in human hepatocytes, that acid phos-phatase is present in GERL and in adjacent coated vesicles. Bertoliniand Hassan 5 reported acid phosphatase activity in the "inner Golgisaccule" in one case of human viral hepatitis, but the published micro-graph strongly suggests that the activity is in GERL.The transport of hydrolases to autophagic vacuoles may involve an-

other route. There appears to be general agreement now that, at leastin hepatocytes, autophagic vacuoles are formed as a result of bits ofcytoplasm becoming surrounded by a portion of SER.50'66 Acid phos-phatase and arylsulfatase 66 have been demonstrated within theenveloping SER. It was suggested that in the case of autophagic vacu-oles, acid hydrolases may be transported directly from the ER."3

Continuities between the membranes of peroxisomes and ER havebeen repeatedly observed, in both rat 3 and mouse liver,67 and also inthis study (Fig 21). Such continuities are interpreted to indicate trans-port of peroxisomal enzymes from the ER cisternae by a direct route.33The close spatial relationship of peroxisomes with SER and glyco-

gen may be relevant to some of the proposed peroxisomal functions-eg,it has been suggested that peroxisomes may have a role in gluconeogen-esis,3 and in the metabolic breakdown of cholesterol.38 However, thesefunctions have not been established. The association between the pres-


ence of urate oxidase in a tisstue and the occurrence of highly-stnrctured cr'%stalloid cores in peroxisomes of that tissue ha-e beenemphasized. 39 Howe-er, cry-stalline cores have been found in thereco-ery- stage of a case of cholestatic jaundice in man.6" WAe have nowobserved noncrv-stalline cores in one normal human li-er, and in casesof cirrhosis, hepatitis and cholestasis (uinpublished). In man, the pres-ence of urate oxidase has not been reported.

Mlitochondria. In contrast to peroxisomes, the ER most closely asso-ciated with the mitochondria is the rough-surfaced variety. The RERand the mitochondria also appear to be closel- related futnctionally.Apart from being an energy source, the mitochondria possess enzymesthat are intimately involved %vith microsomal enz-mes in the formationof triglyceride from free fatty- acids,69 and probably- also in heme sv-n-thesis.`"' -Aminolevulinic acid sv-nthetase is apparentl- sy-nthesized inthe RER, then transferred to the mitochondria.T"

Heterogeneity Within the Hepatic Lobule

Morphologic and biochemical heterogeneity- w-ithin the hepatic lob-ule, amply demonstrated in the rat,1" has also been observed in ourhuman material. The intralobular variations in histochemical stainingfor several rat liver enzy%rmes hav-e been confirnmed b- biochemicalanaly sis of unfixed tissues obtained by- microdissection. The zonaldifferences in various histochemical reactions for enz-me are thusconsidered to reflect real quantitative differences. It is possible, how-ever, that these differences may- also be a result of zonal -ariations inthe stability of enzymes to the maanipulations involved in the histo-chemical demonstration of these enznmes. It should be stressed thatenzyme acti-ities demonstrated by- staining methods represent only- afraction of the enzy-mes present in rico. Studies such as those bN-Janigan" hav-e demonstrated the inhibitor!- effects of -various fixativ-eson enzy%mes and the varing sensitivity- of different enzx-mes to thesame fixative.The full significanice of intralobular heterogeneity- remains to be

elucidated, but surel- it must be related to the phy-siology of the li-erand to changes in disease. Thus, the relative abundance of SER incentrolobular hepatocy-tes may- correlate with higher rates of bilinLibinand drug metabolism. Again, the periportal hepatoc-tes w.ith moreplentiful RER may- be more active in the synthesis of secretor!- pro-teins such as VLDL. In cholestasis, stagnation of bile flows- appears firstand is most pronounced in the centrolobular zone. It w-ould be interest-ing to stud!- the relation of this to the smaller bile canaliculi v-ith theirlower levels of staining for ATPase activity.


Unusual Mitochondria. Crystalline inclusions within mitochondriahave been previously observed in normal livers 72 although they are farmore common in a variety of human liver diseases.73 Their origin is notknown. Optical diffraction studies of the unit cell of these inclusions 73

suggest that its constituents are either phospholipid micelles or somerelatively large protein molecules. Failure of these inclusions to oxidizeDAB suggests that they are not metalloproteins.

Intranuclear Glycogen

This type of nuclear inclusion is common in diabetic patients andhas been reported in diverse disease states.74 Chipps and Duff 74 reportedthe phenomenon in 140 autopsied cases of deaths from pathologiccauses; these inclusions were not seen in uncomplicated traumaticdeaths, suggesting that they are a manifestation of pathology. Althoughthese inclusions have not been previously reported in normal humanhepatocytes, they have been reported in normal hepatocytes of thetadpole.75 Unlike the glycogen enclosed by a double membrane andrepresenting cytoplasmic invaginations into the nuclei,7 the intranuclearglycogen without a limiting membrane 77 is believed to be either a resultof nuclear synthesis of glycogen de novo or migration of glycogen fromthe cytoplasm.

SummaryTwelve normal human biopsies have been studied. Intense alkaline

phosphatase and nucleoside monophosphatase activities characterizethe sinusoidal surface, whereas high levels of nucleoside triphosphatasecharacterize the bile secretory surface. Nucleoside phosphatase activitiesare higher in periportal than centrolobular bile canaliculi. Variablenumbers of previously unrecognized vacuoles (100-150 m[i) are presentin the pericanalicular zone.

Nucleoside diphosphatase and thiamine pyrophosphatase reactionproducts are demonstrated in the ER and in several "inner" Golgisaccules. Periportal cells have more abundant RER but less SER thancentrolobular cells. The ER cisterna is considered as a distributioncenter, transporting materials to various parts of the cell-eg, (1)peroxisomal enzymes to peroxisomes by direct membrane continuity;(2) esterified fatty acids to the Golgi saccules via vesicles that budfrom the ER; (3) acid hydrolases to lysosomes via GERL. It is sug-gested that GERL, as well as the Golgi apparatus, may be involved inthe packaging of very low density lipoprotein (VLDL).

Mitochondria are usually partly surrounded by RER cisternae. Theyare longer and stain more intensely for 3-dihydronicotinamide adenine


dinucleotide reductase activity with tetranitro blue tetrazolium as ac-ceptor in periportal hepatocv tes, but no zonal difference is detected intheir abilitv to oxidize diaminobenzidine (DAB) at pH 6.

Peroxisomes are closelv associated with the smooth ER and oxidizeDAB at pH 9.

References1. Novikoff AB, Essner E: The liver cell: some nexv approaches to its study.

Amer J 'Med 29:102-131, 19602. Novikoff AB: Cell heterogeneitv wvithin the hepatic lobule of the rat: stain-

ing reactions. J Histochem Cvtochem 7:240-244, 19593. Shank RE, Morrison G, Cheng CH, Karl I, Schwartz R: Cell heterogeneitv

within the hepatic lobule (quantitative histochemistry). I Histochem Cvtochem7:237-239, 1959

4. Cachera R, Darnis, F: Examen du foie human au microscope electronique.Sem Hop Paris. 31:2187-2200, 1955

5. Brown DB, Delor CJ, Greider 'M, Frajola 'WJ: The electron microscopy ofhuman liver. Gastroenterologp 32:103-118. 1957

6. Popper H, Schaffner F: Fine structural changes of the liver. Ann Int Med59:674-691, 1963

7. Biava CG: Studies on chclestasis: a re-evaluation of the fine structure ofnormal human bile canaliculi. Lab Invest 13: 840-864, 1964

8. Schaffner F: Morphologic studies on bile secretion. Amer J Dig Dis 10:99-115, 1965

9. Amakawa, T: Electron microscopic studies on lv-sosomes in human hepaticparenchymal cells. J Electron Micr 16:154-168, 1967

10. Baker JR: The structure and chemical composition of the Golgi element.Quart J Micr Sci 85:1-71, 1944

11. Sabatini DD, Bensch K, Barrnett RJ: Cvtochemistrv and electron microscopv:the preservation of cellular ultrastructure and enzvmatic activity by aldehvdefixation. J Cell Biol 17:19-58, 1963

12. Novikoff AB: Lvsosomes in the physiolog- and pathology of cells: Con-tributions of staining methods. Ciba Foundation Symposium on Lvsosomes.Edited by AV'S deReuck, MP Cameron. Boston, Little, Brown and Co, 1963

13. Barka T, Anderson PJ: Histochemical methods for acid phosphatase usinghexazonium pararosanilin as coupler. J Histochem Cvtochem 10:751-753,1962

14. Havashi M, Nakajima Y, Fishman WVH: The cytologic demonstration of -glucuronidase employing naphthol AS-BI glucuronide and hexazonium para-rosanilin: a preliminarv report. J Histochem Cvtochem 12:293-297, 1964

15. Novikoff AB, Essner E, Goldfischer S, Heus M: Nucleoside phosphataseactivities of cvtomembranes. Svmpos Int Soc Cell Biol 1:149-192, 1962

16. Novikoff AB: Membrane bound enzymes, Sixth International Congress ofBiochemistry, New York, VIII-S4:609-610, 1964

1 7. Gomori G: Microscopic Histcchemistry: Principles and Practice. Chicago.University of Chicago Press, 1952, pp 189-194

18. Novikoff AB, Goldfischer S: Nucleosidediphosphatase activity in the Golgi


apparatus and its usefulness for cytological studies. Proc Nat Acad Sci USA47:802-810, 1961

19. Novikoff AB, Sinl WY, Drucker J: Mitochondrial localization of oxidativeenzymes: staining results with two tetrazolium salts. J Biophys Biochem Cytol9:47-61, 1961

20. Novikoff AB, Goldfischer S: Visualization of peroxisomes (microbodies) anidmitochondria with diaminobenzidine. J Histochem Cytochem 17:675-680,1969

21. Smith RE, Farquhar MG: Preparation of nonfrozen sections for electronmicroscopic cytochemistry. RCA Sci Instr News 10: 13, 1965

22. Caulfield JB: Effects of varying the vehicle for OsO, in tissue fixation. JBiophys Biochem Cytol 3:827-830, 1957

23. Lillie RD: Histopathologic Technique and Practical Histochemistry. NewYork, McGraw-Hill Book Co, 1954, p 451

24. Luft JH: Improvements in epoxy resin embedding methods. j Biophys Bio-chem Cytol 9:409-414, 1961

25. Farquhar M, Palade G: Cell junctions in amphibian skin. j Cell Biol 26:263-291, 1965

26. Rappaport AM: Acinar units and the pathophysiology of the liver, TheLiver. Vol 1. Edited by C Rouiller. New York, Academic Press, 1962, pp 265-328

27. Reynolds ES: The use of lead citrate at high pH as an electron-opaque stainin electron microscopy. J Cell Biol 17:208-212, 1963

28. Watson ML: Staining of tissue sections for electron microscopy with heavymetals. J Biophys Biochem Cytol 4:475-478, 1958

29. Sternlieb I: Mitochondrion desmosome complexes in human hepatocytes.Z Zellforsch 93:249-253, 1969

30. Farquhar MG, Palade GE: Junctional cornplexes in various epithelia. J CellBiol 17:375-412, 1963

31. Bruni C, Porter KR: The fine structuire of the parenchymal cell of the nor-mal rat liver: I. General observations. Amer j Path 46:691-735, 1965

32. Loud AV: A quantitative stereological description of the ultrastructure ofnormal rat liver parenchymal cells. j Cell Biol 37:27-46, 1968

33. Novikoff AB, Shin WY: The endoplasmic reticulum in the Golgi zone andits relation to microbodies, Golgi apparatus anid autophagic vacuoles in ratliver cells. J Microscopie 3:187-206, 1964

34. Revnolds, ES: Liver parenchymal cell injurv: III. The nature of calcium-associated electron-opaque masses in rat liver mitochondria following poison-ing with carbon tetrachloride. J Cell Biol 25:53-75, 1965

35. Beard ME, Novikoff AB: Reaction of mitochondria with diaminobenzidine.j Cell Biol 43:12a, 1969, abstr

36. DeDuve C, Baudhuin P: Peroxisomes (microbodies and related particles).Physiol Rev 46:323-357, 1966

37. Goldfischer S, Essner E: Further observations on the peroxidatic activitiesof microbodies (peroxisomes). J Histochem Cytochem 17:681-685, 1969

38. Hruban Z, Rechcigl J Jr: Microbodies and related particles: morphology,biochemistry and physiology. Int Rev Cytol: Suppl 1: 1969

39. Hamilton RL, Regan DM, Gray ME, LeQuire VS: Lipid transport in liver:


I. Electron microscopic identification of very low- density lipoproteins in per-fused rat liver. Lab Invest 16:305-319, 1967

40. Stein 0, Stein Y: Lipid svnthesis, intracellular transport. storage and secre-tion: I. Electron microscopic radioautographic studv of liver after injectionof tritiated palmnitate or glvcerol in fasted and ethanol-treated rats. J Cell Biol33:319-339, 1967

41. Jones AL, Ruderman NB. Evans JB: An electron microscopic study ofhepatic lipoprotein synthesis in the rat following anti-insulin serum, nicotinicacid and puromvcin administration. Proceedings of the American Associationfor the Study of Liver Disease, Chicago, 1968

42. Biempica L, Roheim PS, Kosower NS: Changes in lipids and endoplasmicreticulum of rat hepatocvtes during experimental porphyria. J Cell Biol 39: 18a.1968, abstr

43. 'Mahlev RW, Hamilton RL, LeQuire, V'S: Characterization of lipoproteinparticles isolated from the Golgi apparatus of rat liver. J Lipid Res 10:433-439.1969

44. Novikoff AB: Lvsosomes in nerve cells, The Neuron. Edited by H Hvden.Amsterdam, ElseVier Publishing Co, 1967, p 346

45. Havashi MI: Comparative histochemical localization of lysosomal enzymesin rat tissue. J Histochem Cvtochem 15:83-92. 1967

46. Idem: Histochemical demonstration of N-acetyl-p-glucosaminadase employ-ing naphthol AS-(l N-acetyl-p-glucosaminide as substrate. J Histochem Cvto-chem 13:355-360, 1965

47. Essner E, Novikoff AB: Human hepatocellutlar pigments and lysosomes. jUltrastruct Res 3:374-391, 1960

48. Kerr DNS, Muir AR: A demonstration of the structure and distribution offerritin in the human liver cell. J Ultrastruct Res 3:313-319, 1960

49. Friend DS: Cytochemical staining of multivesictular body and Golgi vesicles.J Cell Biol 41:269-279, 1969

.50. Holtzman E: Lvsosomes in the phxsiology and pathology of neurons, L-so-somes in Biology and Pathology. Vol 1. Edited by IT Dingle, HB Fell. Ams-terdam and London, North Holland Publishing Co, 1969, pp 192-216

51. Wachstein M. Meisel E: Histochemistry of hepatic phosphatases at aphysiologic pH: wNith special reference to the demonstration of bile canaliculi.Amner J Clin Path 27:13-23, 19,57

52. Wachstein 'M: Enzymatic histochemistrv of the liver. Gastroenterolog- 37:525-537, 1959

53. Fawcett DWV: Surface specializations of absorbing cells. j Histochem C-to-chem 13:7.5-91, 1965

54. Levi Aj, Gatmaitan Z, Arias ITM: Two cytoplasmic protein fractions, Y andZ. and their possible role in the hepatic uptake of bilirmbin, sulfobromo-phthalein, ard other anions. J Clin Invest 48:2156-2167, 1969

55. Lester B, Troxler RF: Recent advances in bile pigment metabolism. Gastro-enterolog, 56:143-169, 1969

56. Bemstein LH. Ezzer IB. Garther L, Arias IM: Hepatic intracellular distri-bution of tritium labelled unconjugated and conjugated biliribin in normaland Gunn rats. J Clin Invest 45:1194-1201. 1966

57. Brown WR. Grodskv GM' Carbone JV: Intracelluilar distribution of tritiated


bilirubin during hepatic uptake and excretion. Amer J Physiol 207:1237-1244,1965

58. Alpert S, Mosher M, Shanske A, Alias IM: Multiplicity of hepatic excre-tory functions for organic anions. J Gen Physiol 53:238-277, 1969

59. Palade GE, Siekevitz P, Caro LG: Structure, chemistry and function of thepancreatic exocrine cell, The Exocrine Pancreas. Ciba Foundation Symposium.Edited by AVS deReuck, M Cameron. Boston, Little, Brown and Co, 1962,pp 23-49

60. Friend DS, Farquhar MG: Functions of coated vesicles during protein ab-sorption in the rat vas deferens. J Cell Biol 35:357-376, 1967

61. Miller F, Herzog V: Die Lokalisation von Peroxydase und saurer Phos-phatase in eosinophilen Leukocyten wahrend der Reifung. Z Zellforsch 97:84-110, 1969

62. Shin WY, Ma MH, Quintana N, Novikoff AB: Organelle interrelations inthyroid epithelial cells. Proceedings of the Seventh International Congresson Electron Microscopy, Grenoble, 1970

63. Margolis S: Structure of very low and low density lipoproteins, Structuraland Functional Aspects of Lipoproteins in Living Systems. Edited by E Tria,AM Scann. New York, Academic Press, 1969, p 388

64. Favard P: The Golgi apparatus, Handbook of Molecular Cytology. Editedby A Lima-de-Faria. Amsterdam and London, North Holland Publishing Co,1969, pp 1130-1155

65. Bertolini B, Hassan G: Acid phosphatase associated with the Golgi appara-tus in human liver cells. J Cell Biol 32:216-219, 1967

66. Arstila AU, Trump BF: Studies on cellular autophagocytosis: the formationof autophagic vacuoles in the liver after glucagon administration. Amer J Path53:687-733, 1968

67. Essner E: Endoplasmic reticulum and the origin of microbodies in fetalmouse liver. Lab Invest 17:71-87, 1967

68. Biempica L: Human hepatic microbodies with crystalloid cores. J Cell Biol29:383-386, 1966

69. Favarger P: The liver and lipid metabolism, The Liver. Vol 1.26 pp 549-60470. Scholnick PL, Hammaker LE, Marver HS: Soluble hepatic &-aminolevulinic

acid synthetase: end product inhibition of the partially purified enzyme. ProcNY Acad Sci 63:65-70, 1969

71. janigan, DT: Tissue enzyme fixation studies: 1. The effects of aldehyde fixa-tion on P-glucuronidase, f3-galactosidase, N-acetyl-3-glucosaminidase and f3-glucosidase in tissue blocks. Lab Invest 13:1038-1050, 1964

72. Willis EJ: Crystalline structures in the mitochondria of normal human liverparenchymal cells. J Cell Biol 24:511-514, 1965

73. Sternlieb I, Berger JE: Optical difraction studies of crystalline structures inelectron micrographs. J Cell Biol 43:448-455, 1969

74. Chipps HD, Duff GL: Glycogen infiltration of the liver cell nuclei. AmerJ Path 18:645-659, 1942

75. Himes MM, Pollister AW: Symposium: Synthetic process in the cell nucleus:V. Glycogen accumulation in the nucleus. j Histochem Cytochem 10: 175-185,1962

76. Kleinfeld RG, Greider MH, Frajola WJ: Electron microscopy of intranuclearinclusions found in human and rat liver parenchymal cells. J Biophys BiochemCytol: Suppl 2:435-438, 1956


. Sparrow \VT, Ashworth CT: Electroni microscopy- of nuclear glycogenosis.Arch Path 80:84-90, 1965

ADDEN-DUM: A brief note appeared after this paper was submitted, reporting the oc-currence of marginal plates in patients wvith Wilson's disease and even in normal subjects(,New Eng J Med 283:1290, 1970).

The authors wish to thank M%rs. S. Biempica, M\rs. R. Dominitz, MIiss G. MNitchell andMIr. C. Davis for their excellent technical assistance and microscopic preparations, and Mtr.J. Godrich for preparations of the photographs. We also wvish to thank Drs. A. B. N-ovikoff.Sidney Goldfischer and Mtargaret Beard for frequent discussions during the course of this'Work and for their critical reviewv of the manuscript.

Legends for All FiguresFig 1.-Nucleoside phosphatase, with AMP as substrate. Incubated for 45 minutes.Sinusoidal surface staining (short arrow) is stronger than bile canalicular staining(long arrow). No intralobular difference in sinusoidal staining is seen. PT indicatesportal tract; and CV, central vein (fixed in 2.5% glutaraldehyde in cacodylate buffer;A, x 125; B, x 400).

Fig 2.-Nucleoside phosphatase with ATP as substrate. Incubated for 45 minutes.There is intense bile canalicular staining in periportal zone (PT) but inconstant andweaker staining in centrolobular zone (CV). No sinusoidal activity is evident. Fixed in2.5% glutaraldehyde in cacodylate buffer. (* 125). B.-Area in periportal zone athigher magnification. x 400.

Fig 3A.-Periportal hepatocytes. Complex microvilli are present on sinusoidal surface(S). Plasma membranes on either side of the bile canaliculus (BC) are devoid ofmicrovilli (see also Fig 3B and 12). On lateral surfaces where bile canaliculus is ab-sent, plasma membrane is provided with short interdigitating microvilli (arrows). Mito-chondria are numerous and appear oval or oblong in shape. RER is located in closeproximity to mitochondria. N indicates nuclei; R, erythrocytes; K, Kupffer cell; L, lipo-fuscin (fixed in Veronal-buffered 2% OSO4, X 6000). B.-Many short, nonbranch-ing microvilli (arrows) are seen on lateral surfaces of these two liver cells (fixed inVeronal-buffered 2% 0504, x 30,000).

Fig 4.-Sinusoidal surface. Microvilli sectioned in various directions project intospace of Disse (D) between hepatocyte and endothelial cell (E). Arrows point to bifur-cation of microvilli. Coated vesicles (V) are close to sinusoidal surface (phosphate-buffered 2% OsO, x 34,000).

Fig 5.-Desmosome-mitochondria complex. Two adjacent plasma membranes of des-mosome are strictly parallel and are separated from each other by distinct intercel-lular space. Fine fibrils are located between cytoplasmic plaques (arrows) and mito-chondria on each side (fixed in phosphate-buffered 2% OSO4, x 60,000).

Fig 6.-Bile canaliculus. At tight junction (TJ), external leaflets of adjacent plasmamembranes are fused and appear as single line. Zone of electron-dense amorphousmaterial extends along tight junction and intermediate junction (IJ). Desmosomes arelocated further distally (not included). Minute vesicles are present in ectoplasm sur-rounding bile canaliculus (arrow heads) (fixed in phosphate-buffered 2% OSO4 andrinsed for 1 hour in uranyl acetate before dehydration and embedding, x 60,000).

Fig 7.-Bile canaliculus. Relatively round electron-lucent vacuoles (v) are present inpericanalicular zone of some bile canaliculi. They are different from vesicles of SER,


which are irregular and contain faintly electron-dense material. Arrow heads indicateminute vesicles often found in pericanalicular ectoplasm. G indicates Golgi apparatus(fixed in phosphate-buffered 2% OS04, x 24,000).

Fig 8.-Nucleus. Chromatin clumps (C) are present along inner surface of nuclearenvelope except at nuclear pores (arrows), in association with nucleolus (NU) and inpatches throughout nucleoplasm (fixed in phosphate-buffered 2% OS04, rinsed inuranyl acetate before dehydration and embedding, x 12,000).

Fig 9.-Nuclear pore (NP) formed by fusion of outer and inner nuclear membranes(fixed and prepared as in Fig 8, x 126,000).

Fig 10.-Intranuclear glycogen. Glycogen particles are of monoparticulate type-ie,they are not arranged in form of rosettes characteristic of cytoplasmic glycogen. Fi-brillar ring (arrows) surrounds smaller collection of glycogen, which is more tightlybound than that free in the nucleoplasm (fixed in 3% glutaraldehyde in cacodylatebuffer, followed by phosphate-buffered 1% OS04, X 16,500).

Fig IIA.-Nucleoside diphosphatase activity with IDP as substrate. Incubated for 40minutes. Bile canaliculi (BC) and sinusoids (S) are darkly stained, indicating presenceof nucleoside phosphatase in microvilli. Thread-like staining in hepatocyte cytoplasmrepresents ER activity (arrows) (fixed in 2.5% glutaraldehyde in cacodylate buffer for2 hours, x 400). B.-Nucleoside diphosphatase activity with IDP as substrate.Incubated for 65 minutes. Reaction product is seen in ER cisternae and in nuclear en-velope (NE) (fixed in 2.5% glutaraldehyde in cacodylate buffer for 2 hours, x 38,500).

Fig 12A.-Centrolobular hepatocytes. Most mitochondria appear round or oval. RERis seen mainly near sinusoid (single arrows), nucleus (N), Golgi apparatus (G) andmitochondria. Two of peroxisomes contain nucleoids (double arrows). Autophagic vac-uole (AV) is present near nucleus while several lipofuscin granules (L) are in peri-canalicular area. Nuclei contain dispersed chromatin (fixed in Veronal-buffered 2%OS04, X 12,500). B.-Enlargement of one of nucleoid-containing peroxisomes(double arrows). Close proximity of RER to mitochondria is also shown (x 26,000).

Fig 13A.-Periportal hepatocytes. Elongated mitochondria are more numerous andRER is more plentiful here than in centrolobular cells. Dilated Golgi saccules (G) areevident. Nuclear chromatin granules (C) occur in clumps at nuclear periphery. SERappears as irregularly shaped vesicles between glycogen rosettes. BC indicates bilecanaliculus; S, sinusoid (fixed in phosphate-buffered 2% OSO4, x 12,500). B.-In glutaraldehyde-fixed tissues, SER sometimes appears dilated and shows invagina-tions of membrane. On cross sections, invaginations appear as separate vesicles (ar-rows) (x 60,000).

Fig 14.-Nicotinamide dinucleotide TNBT-reductase. Incubated in NADH2-TNBT for 40minutes. Staining is somewhat less intense in centrolobular zone (CV) than in peri-portal zone (PT) (fixed in 2.5% glutaraldehyde in cacodylate buffer, x 125).

Fig 15.-Diaminobenzidine oxidation in mitochondria. Incubated in DAB medium, pH6.0, for 90 minutes. Reaction product is present in mitochondrial cristae and in innerand outer mitochondrial membranes (see inset). No reaction is evident in peroxisomesand ER (fixed in 3% glutaraldehyde in cacodylate buffer, postfixed in phosphate-buffered 2% OSO4, and rinsed in uranyl acetate before dehydration and embedding,x 11,500; inset, x 46,000).

Fig 16.-DAB oxidation reaction in mitochondria. Incubated in DAB medium, pH 9,for 90 minutes for visualizing peroxisomes. Cristae of some mitochondria contain re-action product (fixed in 3% glutaraldehyde in cacodylate buffer, postfixed in phos-phate-buffered 2% OSO0 and rinsed in uranyl acetate before dehydration and embed-ding, x 100,000).

Fig 17.-Crystalline inclusions in mitochondria. The inclusions are cut at differentangles in Ml and M2 (fixed in phosphate-buffered 1% OS04, x 39,500).

Fig 18.-DAB oxidation reaction in mitochondria. Crystalline inclusions are not stained


in DAB medium in contrast to mitochondrial cristae, which are stained. Intramito-chondrial granules also appear stained (arrows) (fixed in 3% glutaraldehyde in caco-dylate buffer, incubated in DAB medium at pH 6 for 100 minutes and postfixed inphosphate-buffered 1% OsO,, x 51,000).

Fig 19.-"Curling" of cristae (arrows) is evident in several mitochondria (fixed inphosphate-buffered 2% OSO4, x 25,500).

Fig 20.-DAB oxidation in peroxisomes. Incubated in DAB medium, pH 9, for 90 min-utes. All peroxisomes that are present contain flocculent reaction product (fixed in 3%glutaraldehyde and postfixed in phosphate-buffered 1% OsO, unstained, x 26,000).

Fig 21A.-Continuity between RER and SER is shown at arrow. Peroxisome (P) showsarea of electron-opaque condensation of matrix. Note also accumulation of RER cis-temae along sinusoidal surface (S) (fixed in phosphate-buffered 2% OSO4, x 48,500).B.-Peroxisome showing continuity with SER (fixed in phosphate-buffered 2% OSO4and rinsed in uranyl acetate before dehydration and embedding, x 87,000).

Fig 22.-Portion of hepatocyte adjacent to central vein is shown here with peroxi-somes outnumbering mitochondria (fixed in phosphate-buffered 2% OSO4, x 10,000).

Fig 23.-Thiamine pyrophosphatase. Incubated in TPP medium for 110 minutes.Staining is seen in Golgi apparatus (arrows) of several cells. Bile canaliculi alongwhich Golgi apparatus are oriented are also stained (fixed in 4% formaldehyde incacodylate buffer, x 520).

Fig 24.-Curved Golgi saccules are parallel to each other, forming stack with its con-vex surface toward bile canaliculus (BC). Many vesicles are present on both concaveand convex surfaces of Golgi apparatus. Arrow 1 points to connection between vesicleand outer Golgi saccule. Arrow 2 indicates vesicle that appears as if budding fromRER cistema. Structure with smooth membrane adjacent to concave surface of Golgiapparatus is interpreted as GERL (GE). Short segment of GERL membrane is coated(double arrows). Arrow 3 points to area of transition between RER and smooth mem-brane, which is probably part of GERL. VLDL particles are present in cistema of GERLand in Golgi saccules (GS) (fixed in 3% glutaraldehyde in cacodylate buffer, postfixedin phosphate-buffered 2% 0504, X 26,000).

Fig 25.-Golgi region. Parallel arrangement of Golgi saccules (GS) is seen. Vacuoles(long arrows) and dilated portions of Golgi saccules (short arrows) contain dense, ir-regularly shaped particles considered to be VLDL. Two muftivesicular bodies (MVB)are also seen (fixed in phosphate-buffered 2% OsO, x 20,000).

Fig 26.Golgi apparatus. Numerous smooth vesicles are seen along convex surfaceof Golgi apparatus. VLDL particles are seen in structure with smooth membrane,which is probably GERL (GE). and in partly coated vacuole (CV). Two coated vesicles(arrows) are also seen (fixed in phosphate-buffered 2% Os04 and rinsed in uranylacetate before dehydration and embedding, x 60,000).

Fig 27.-Thiamine pyrophosphatase. Incubated in TPP medium for 210 minutes. Re-action product is concentrated in Golgi saccules on concave surface of Golgi appa-ratus. Vacuole (arrow) whose content of VLDL is partially extracted by preparatoryprocess is devoid of reaction product (fixed in 3% glutaraldehyde in cacodylate bufferfor 2 hours and postfixed in phosphate-buffered 2% OsO after incubation, rinsed inuranyl acetate before dehydration and embedding, x 60,000).

Fig 28-Nucleoside diphosphatase. Incubated in IDP medium for 65 minutes. Reac-tion product is present in the two, perhaps three, saccules on concave side of Golgiapparatus. Note also clumps of reaction product on microvilli of bile canaliculus (BC)(fixed in 4% paraformaldehyde in cacodylate buffer and postfixed in phosphate-buffered 2% SOS4 after incubation, x 30,000).

Fig 29.-Acid phosphatase reaction. Incubated in CMP for 15 minutes. Lysosomaistaining of periportal hepatocytes (PT) is somewhat more intense than in hepatocytesof centrolobular zone (CV) (fixed in 3% glutaraldehyde in cacodylate buffer, x 125)


Fig 30.-Acid phosphatase. Higher magnification of periportal zone. Small discretepericanalicular lysosomes are evident (arrows). Kupffer cell lysosomes (K) are largerand less numerous (x 440).

Fig 31.-Glucosaminidase. Incubated for 120 minutes. Lysosomal staining is not seenin hepatocytes but is present in a few Kupffer cells (arrows) (fixed in 3% glutaral-dehyde in cacodylate buffer, x 440).

Fig 32.-Near bile canaliculus (left upper corner) may be seen autophagic vacuoles(AV,, AV, and RB) and lipofuscin granule (L). AV, has a double limiting membraneand contains recognizable mitochondria, glycogen and ER. AV, and RB have singlelimiting membranes and may represent more advanced stages of autophagy (fixed inphosphate-buffered 1% OSO4, X 22,000).

Fig 33.-Pericanalicular region. Multivesicular body (MVB), delimited by single mem-brane, contains small vesicles and homogeneous material. Autophagic vacuole containssmooth vesicles identified as SER (fixed in phosphate-buffered 2% OsO, x 30,000).

Fig 34.-Multivesicular body. Vesicles within MVB resemble those adjacent to it (ar-row). Pseudopod-like projections resemble nearby tubular structures (T) (fixed inphosphate-buffered 2% OSO4, x 60,000).

Fig 35.-AV, and AVll and inset are autophagic vacuoles, with varying degrees of ac-cumulation of ferritin particles. Free ferritin particles are also present in cytoplasm(arrows) (fixed in phosphate-buffered 2% OS04, unstained, x 40,500).

Fig 36.-Acid phosphatase. Incubated in CMP medium for 40 minutes. Reaction prod-uct is seen in smooth tubular system (long arrow) lacking parallel arrangement ofadjacent Golgi saccules and separated from them by vesicles (short arrows). It is con-sidered to be GERL. Reaction product is also present in two nearby vesicles (V), andin autophagic vacuole (AV) (fixed in 3% glutaraldehyde in cacodylate buffer and post-fixed in phosphate-buffered 2% Os04, x 39,000).

Fig 37.-Acid phosphatase. Incubated in CMP for 60 minutes. Reaction product islocalized in matrix between vesicles in multivesicular body and in lipofuscin granule(L) (fixed as in Fig 36, x 78,000).


[ lUustrations follow ]

I B

2B

1A

2A

R s~~~

0fno tatAo _ -,s _

,,.-7O-0iliiL; e>- - -oss

, ,. ;; ^

,>:'rs x 's, .;

_

J7

3A

3B

tT- X~

4

6

5

7

8

9109

IV pis-.. ~4,A

I IA

I

- e' -n

r,t m- (..-.-'

. :.Z.

i m

, a

.jj.&A*' rtk.

&

"I -3~~

i)4:A\ ."

12A

1 2 B

JA

17"

6.7

Al-

lo

71,

I

I

It

b

i

14

I) IC

13A

13B

I

15 '

a.. !

16

18

17

1 9

I-

20

21A

21B

...i

"I

swo. .0

al.

22

2423

25

27

26

28

30

29

31

32 33

ofp..W-w<'Le~'Iw$f-

34

36

a- 35

,)

,i

I

.1

'

37

._c'W. W.4;.--,

P,*. .-%

V. " '1%i"

.0lb

1- rlAl

.1

The Normal Human Liver Cell - NCBI

Documents

Transcript of The Normal Human Liver Cell - NCBI