J Clin Pathol 1982;35:1078-1085

Micronodular adrenal disease: a light and electronmicroscopic studyPS HASLETON, HH ALI, C ANFIELD, CG BEARDWELL, S SHALET

From the Departments of Pathology and Endocrinology, Withington Hospital, Manchester

SUMMARY A case of Cushing's syndrome due to micronodular adrenal disease in a 17-year-oldgirl is presented. The adrenals showed both black and yellow nodules. Histologically the cellscontained lipofuschin and either had a clear cytoplasm or an eosinophilic cytoplasm with a promi-nent nucleus. Lymphocytes were a prominent feature. No cells of the zona reticularis were identi-fied. The cell of origin of these nodules appeared to be from the inner layer of the zona

fasciculata. We postulate that the disease is caused by an abnormality in the migration and ultimatedestruction of cells from the zona fasciculata to the zona reticularis with a build up of cells at theinterface zone.

Micronodular adrenal disease (primary adrenal corti-cal dysplasia) is an uncommon cause of Cushing'ssyndrome. In a recent monograph on adrenal dis-easel micronodular disease is not mentioned. Pre-vious reports accurately describe the macroscopicand light microscopic features but are unclear as tothe origin of the cells in the nodules. We describebelow a case of primary adrenal cortical dysplasiawith electron microscopic findings to determine thecell type responsible for the adrenal nodule forma-tion.

Case report

A 17-year-old girl was admitted to hospital becauseof hirsuitism and secondary amenorrhoea. Her earlygrowth and development had been normal.Menarche occurred at the age of 12 but periodsstopped one year later at which time she developedthe classical features of Cushing's syndrome whichprogressed steadily over the next four years.Biochemical investigations showed a greatly raisedbasal 24-hour urinary excretion of 11-hydroxycorticosteroids, 1 7-oxogenic steroids and17-oxosteroids with an absent diurnal variation ofplasma cortisol, thus confirming the clinical diagnosisof Cushing's syndrome.The plasma ACTH level was undetectable on sev-

eral occasions and the urinary excretion of 17-oxogenic steroids was unaltered by eithermetyrapone or dexamethasone administration.An x-ray examination of the pituitary fossa, dorsal

and lumbar spine was completely normal. Abdominal

Accepted for publication 8 February 1982

CT scan showed that the right adrenal glandappeared larger than the left adrenal gland. Scinti-graphy with radioisotope-labelled cholesterol showeduptake only in the region of the right adrenal. In viewof the radiological and biochemical findings it wasthought that she had a right-sided adrenal tumour. Aright adrenalectomy was performed. The glandappeared normal in size and showed many micro-nodules but no adenoma. The provisional diagnosiswas revised to that of micronodular adrenal diseaseand since this is usually bilateral, it was thoughtadvisable to remove the remaining adrenal. After thesecond operation the clinical features of Cushing'ssyndrome resolved and regular menses returned.

PATHOLOGYThe right adrenal weighed 8-2 g, the left 5.3 g. Theappearance of both glands was similar. There werenumerous nodules, some black and others yellow setin a yellow cortex. The largest nodule measured1 x 0-5 cm. The right adrenal was fixed in Carson'sfluid. Small blocks (1 mm3) were taken from the leftadrenal and fixed in 4% glutaraldehyde in Sorenson'sphosphate buffer at 4°C. The blocks were post-fixedin 1% phosphate-buffered osmium tetroxide. Thematerial was embedded in Epon resin. Sections (1gm) were cut and stained with toluidine blue. Thinsections were cut on an LKB IV ultramicrotome,stained with uranyl acetate and lead citrate andexamined on an AEI EM810 electron microscope.

Blocks from the right adrenal were first washed incacodylate buffer, post-fixed in osmium tetroxideand embedded in Epon resin as above. Blocks weretaken from both the yellow and black areas.On light microscopy the presence of nodularity

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Fig. 1 Low powerphotomicrograph showing thetypical nodular appearance oftheadrenal. Haematoxylin and eosin x120.

was confirmed (Fig. 1). The zona glomerulosa wascompressed in areas but in other fields it dippeddown between the nodules and appeared thickerthan normal. The zona fasciculata was sparse butgroups of cells with a clear cytoplasm lying outsidethe nodules were identified. No cells from the zonareticularis were found in the non-nodular adrenal tis-sue.The nodules which were of two types composed

the majority of the adrenal glands. The most promi-nent type of nodule consisted of large cells with aneosinophilic cytoplasm, a large nucleus that waseither open and vesicular or pyknotic (Fig. 2). Othernodules consisted of large clear cells. Some of thenodules consisted of both clear and eosinophilic cells.The reticulin stain showed the nodular pattern well.The eosinophilic cells contained a dark brown pig-ment that was not haemosiderin or melanin butstained positively with a prolonged Ziehl-Neelsenstain proving the presence of lipofuschin. Betweenthe nodules were many dilated capillaries. Somenodules were present outside the capsule, in theperiadrenal fat.

In relation to some nodules were foci of lympho-cytes (Fig. 2). The cells showed no evidence ofnecrosis in these areas and no giant cells were seen.

Prominent phaeochromocytes were present, some-times as isolated cells but otherwise the medulla wasnormal. Ganglion cells were seen in the adrenal fat,in the capsule and inside the cortex in one instance.Hypertensive arterioles were present in the peri-adrenal fat.

ELECTRON MICROSCOPYBlocks were taken from both black and yellow foci.The cells had microvilli, a convoluted plasma mem-brane and large prominent mitochondria. The mito-chondria had a tubulovesicular internal structure(Fig. 3). Many dense black homogeneous lysosomeswere present (Fig. 4). In some cells the mitochondriahad a mixture of dark and pale circular areas as wellas lamellar structures inside them. Such mitochondriamay be undergoing lysis. Myelin figures were alsoseen (Fig. 5). Many free and membrane-attachedribosomes were present. Foci of black material, notbound by any membrane, were present. In other cellsthe lipid had dissolved out leaving clear foci. Roughendoplasmic reticulum was present in many areasforming parallel stacks. The nuclei had small inden-tations but were otherwise unremarkable. Such cellsare typical of the interface region of the zona fas-ciculata.

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Fig. 2 Part ofa nodule with thezona glomerulosa in the top lefthand corner. Most ofthe cells havean eosinophilic cytoplasm thoughcells with a clear cytoplasm arepresent. Foci oflymphocytes(arrows) are present. Haematoxylinand eosin x 211.

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Fig. 3 Cell ofinner zonafasciculata with numerousmitochondria (M), microvilli(arrow). Uranyl acetate and leadcitrate x 7000. The inset showsmitochondria with a tubulovesicularinternal structure. Uranyl acetateand lead citrate x 28 800.

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Fig. 4 Area from a black nodule-fi showing dense lysosomes abutting a

nucleus. Uranyl acetate and leadcitrate x 20 000.

Fig. 5 Mitochondria with both dark and pale circular areasas well as a myelin figure (arrow). Uranvl acetate and leadcitrate x 12 600.

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Fig. 6 Part ofa cellfrom a yellownodule with many mitochondriaand much lipid. Uranyl acetate andlead citrate x 18 500.

Fig. 7 Cells ofthe zonaglomerulosa (G) with a largenucleus, regular cell border andfewmitochondria (M). The capsule (C)is seen at the top ofthe picture.Uranyl acetate and lead citrate x5000.

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Micronodular adrenal disease: a light and electron microscopic study

Blocks from the yellow nodules showed a similarappearance to the black areas but there were moreclear foci, some of which presumably representedfat that had dissolved out (Fig. 6). Other areas wereprobably endoplasmic reticulum. The mitochondriawere prominent and these also had a tubulovesicularinternal structure. Thus these cells also showed thecharacteristics of the zona fasciculata from the inter-face zone.

Cells of the zona glomerulosa were identified (Fig.7) with a larger nuclear/cytoplasmic ratio than thecells in the zona fasciculata. The basement mem-brane was not crenated. Mitochondria had a regularcristate internal structure and the cells containedosmiophilic lipid. Few lysosomes were present.No cells from the zona reticularis were seen either

inside or outside the nodules.

Discussion

Cushing's syndrome is caused by iatrogenic admini-stration of steroids, pituitary or adrenal diseases, orless commonly, is due to ectopic secretion of ACTHby non-endocrine tumours. In this patient all thebiochemical and radiological findings suggested anadrenal origin.

Histologically the appearance was typical of micro-nodular adrenal disease. There were nodules ofvarying sizes. The nodules consisted either of largeclear cells or of cells having an eosinophilic cytoplasmwith a dark brown pigment. Lymphocytes wereprominent in areas.The histological picture has been described pre-

viously.2' 3 Characteristically, both adrenals areinvolved though it should be noted that usually theadrenal is normal in weight. The upper limit of thenormal surgically resected adrenal is 4 g with an SDof 0-8() g.4 In our case both adrenals were heavierthan normal, the right gland weighing 8*2 g and theleft 5.3 g. In Meador's case2 the combined weightwas 9*5 g, Ruder described two cases.3 In case 1 theleft gland weighed 3-5 g and the combined weight incase 2 was 4.0 g, the right adrenal weighing only0 9 g.

Characteristically, the disease affects children inthe second decade of life, the age range in Ruder'sseries being 14-37 yr. Females tend to be affectedmore commonly than males in a ratio of 3:1 asdetermined from the cases quoted by Ruder.3 Onecase of an infant presenting soon after birth withmicronodular adrenal disease has been described.5Two cases of familial Cushing's syndrome have beendocumented.6 However, though the adrenals hadnumerous micronodules in Klevit's case5, there is nodescription in the paper of either a black colourationof the adrenal or evidence of many lysosomes on

electron microscopy. Similarly, in the case reportedby Donaldson et all there is no illustration of thehistology or electron microscopy. We query, there-fore, whether this is the same disease entity as we aredescribing. The brother of our case was investigatedfor Cushing's syndrome as he was obese. However,there was no biochemical or sound clinical evidenceof Cushing's syndrome.

In the previous studies there has been a lack ofagreement as to the origin of cells in the nodules.Meador et a12 thought the cells were derived fromzona reticularis as they were separated from the cap-sule by well defined fasciculata and glomerulosalayers. Ruder3 stated that the zone of origin of thenodules was unclear. In order to clarify this point weundertook electron microscopy from both yellow andblack adrenal tissue.The cells were arranged in groups and had micro-

villi, a convoluted plasma membrane and promi-nent large tubulovesicular mitochondria. Many lyso-somes were present in the cytoplasm of the cell.These features are those of the zona fasciculataoccuring at the interface zone7 with the zonareticularis. The zona glomerulosa has no microvilliand in the zona reticularis microvilli are patchy. Simi-larly, the mitochondria in the glomerulosa, thoughnumerous, have regular cristae and the mitochondriain the reticularis have an empty appearance with afew peripheral tubules. Lipid vacuoles were presentin some cells and such material is not usually presentin the zona reticularis. Mitochondrial granules arefrequent in this case and are characteristic of theinterface zone of the zona fasciculata. Such granulesare present in the zona reticularis but are fewer innumber than in the fasciculata. It is of interest thatthe interface zone of the zona fasciculata is the areaof the adrenal believed to be the target site for theearly action of ACTH.7A constant feature of micronodular adrenal dis-

ease is the presence of many lysosomes. These inter-related membrane-limited elements form an intracel-lular digestive system for cells. They degrade bothintracellular and extracellular substances. Symington7felt it was probable that pigment dissolved in the lipidstore of clear cells ultimately ended up in a residualbody derived from lysosomes.The tubulovesicular appearance of the mitochon-

dria seen in our case is nearly always seen in cellsproducing steroids.7 It has been suggested that thevesicles are discharged either into the cytoplasm ofthe cell8 or open onto the mitochondrial surface todischarge their contents-for example, corticosteroneor cortisol. The dense granules in the mitochondriaare possibly the site of calcium or other ion accumu-lation. The exact role of calcium in steroid synthesisis not known.

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The fact that the zona fasciculata is the abnormalzone in micronodular adrenal disease fits with cur-

rent concepts of glucocorticoids being produced bythe two inner zones of the adrenal cortex. It isthought that the zona reticularis and fasciculata are a

single functional zone with the fasciculata comprisinga ready reserve capable of being activated intosteroid production.

In the above case there was little difference elec-tron microscopically between the yellow and blacknodules, the former having more clear spaces whichcontained lipid.The final question is what is the aetiology of the

condition? Meador et a12 suggested that it mightresult either from long continued ACTH over-production or from a primary adrenal disorder. Theformer seems unlikely for several reasons. Mostcases of micronodular dysplasia present in the seconddecade of life while some are certainly congenital.Thus the age distribution is quite different fromCushing's syndrome secondary to ACTH hypersecre-tion of pituitary or ectopic origin. In our case, as in allthe others where the measurement has been made,ACTH was undetectable in the patient's serum andneither metyrapone nor dexamethasone alteredsteroid secretion, results indicating complete adrenalautonomy. There is no doubt that nodularity candevelop in bilaterally hyperplastic adrenals associ-ated with prolonged, excessive ACTH secretion, andit is possible that some of these nodules could even-tually become autonomous and lead to partial or

complete pituitary suppression.9 However, it wouldseem likely that this is a different condition from thatdescribed above with a different histological pictureof nodules occurring in a hyperplastic cortex and,usually, considerable adrenal enlargement and a dif-ferent pattern of biochemical tests. In micronodulardysplasia both adrenals are usually of normal or onlyslightly increased weight.An intriguing feature of the present study is the

absence of zona reticularis cells both from the nodu-lar and the non-nodular areas of the adrenal. Thoughthere is obviously a large sampling error, no suchcells were seen on electron microscopy. This, we

feel, must have some bearing on the aetiology of thedisease. It has been proposed that the adrenocorticalcells arise at the periphery, migrate towards thecentre and die at the border with the zona reticularisand medulla.'0Wright" studying the prepubertal rat, which has a

histological picture similar to the human, also feltthat there was an inward migration of cells originat-ing in the outer regions of the cortex. He used label-ling indices and studied the lengths of various com-ponents of the cell cycle in prepubertal rat adrenals.It is interesting in the present study that there are

Hasleton, Ali, Anfield, Beardwell, Shalet

many lysosomes in the nodules. Lysosomal enzymesare important in intracellular degradation. Of furtherinterest is the fact that some authors have used thepresence of lipofuschin pigment as an indication ofsenescence in cells.'2 Certainly in other parts of thebody, such as heart and liver, the presence of lipofus-chin is associated with cells showing regressivechange such as atrophy accompanying old age orchronic injury. This may be taken as further proofthat the elderly cells come to reside at the border ofthe zona fasciculata and reticularis.

Similarly, centripetal flow of cells has been pro-posed to explain regeneration after enucleation ofthe gland.'3 However, most authors believe that thezona glomerulosa is independent of pituitary controlwhile the two inner zones depend on ACTH.

In conclusion, part of the aetiology of nodularadrenal disease could be explained on the centripetalflow theory." If the adrenal cells migrate centrallythere must be some block in the development intothe zona reticularis. This will lead to a build up ofcells at the interface zone which form nodules. Thesecells at the interface zone of the fasciculata with thereticularis normally are capable of secreting bothandrogens and corticosteroids and thus behave func-tionally in a similar way to the zona reticularis. How-ever, in micronodular adrenal disease, there is afurther change in that the accumulating cells also losetheir dependence on ACTH stimulation and developautonomous function. As a result it seems likely thatthey will produce cortisol continuously whereas thenormal adrenal is active only intermittently. This mayexplain why adrenals of almost normal weight areable to produce abnormally large amounts of cortisol.The fundamental cause of these changes, though,remains unknown.

References

1 James VHT. The adrenal gland. New York:Raven Press, 1979.2 Meador CK, Bowdoin B, Crawford Owen Jnr, Farmer AT. Prim-

ary adrenocortical nodular dysplasia: a rare cause of Cushing'ssyndrome. J Clin Endocrinol 1967;27:1255-63.

3 Ruder HJ, Loriaux DL, Lipsett MB. Severe osteopenia in youngadults associated with Cushing's syndrome due to micronodularadrenal disease. J Clin Endocrinol Metab 1974;39:1138-47.

4 Studzinski GP, Hay DCF, Symington T. Observations on theweight of the human adrenal gland and effect of preparations ofcorticotrophin of different purity in the weight and morphologyof the human adrenal gland. J Ctin Endocrinol Metab1963;23:248-54.

Klevit HD, Campbell RA, Blair HR, Bongiovanni AM. Cushing'ssyndrome with nodular adrenal hyperplasia in infancy. JPediatr 1966;68:912-20.

Donaldson MDC, Grant DB, O'Hare MJ, Shackleton CHL.Familial congenital Cushing's syndrome due to bilateral nodularadrenal hyperplasia. Clin Endocrinol 1981;14:519-26.

7Symington T. Functional pathology of the human adrenal gland.Edinburgh and London: Livingstone, 1969.

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' Sabatini DD, De Robertis EDP. Submicroscopic study of thepituitary action in the adrenocortex of the rat. Endocrinology1962;70:390-406.

' Anderson DC, Child DF, Sutcliffe CH, Buckley CH, Davies D,Longson D. Cushing's syndrome, nodular adrenal hyperplasiaand virilising carcinoma. Clin Endocrinol 1978;9:1-14.

Salmon TN, Zwemer RL. A study of the life history of cortico-adrenal gland cells of the rat by means of trypan blueinjections. Anat Record 1941 ;80:421-9.

Wright NA. Cell proliferation in the prepubertal male rat adrenalcortex; an autoradiographic study. J Endocrinol1971;49:599-609.

I Bennett HS. The life history and secretion of the cells of theadrenal cortex in the cat. Am J Anat 1940;67:151-227.

u Skelton FR. Adrenal regeneration and adrenal regenerationhypertension. Physiol Rev 1959;39:162-82.

Requests for reprints to: Dr PS Hasleton, ConsultantPathologist, Wythenshawe Hospital, Southmoor Road,Manchester M23 9LT, England.

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