Farber's lipogranulomatosis

Farber’s Report

ofa

Lipogranulomatosis*

Case and Demonstration

of an Excess of

Free Ceramide and Ganglioside

Boston. Massachusetts The tenth case of Farber’s lipogranulomatosis is reported. The clinical and pathologic features closely resemble those previously described except that in this patient the macula was also involved, there was nerve cell loss and neuronal storage in the cerebral cortex, and there was a strong family history of rheumatoid arthritis. The foam cells within the granulomas as well as certain of the neurons COW tained periodic acid-Schiff positive cytoplasmic material with the histochemical properties of an acidic glycolipid. Biochemical studies demonstrated an excess of gangliosides, roughly in proportion to the accumulation of the periodic acidSchiff-positive material. The excess gangliosides wcrc found to consist mainly of hematoside, the visceral ganglioside. Lymph nodes, liver, kidney and lung contained a ten- to sixtyfold excess 01 free ceramide, and similar very high levels of free ceramide were present in a subcutaneous nodule. In rats, subcutaneous injection of ceramide produced granulomas which resembled those found in the patients with the lipogranulomatosis syndrome. It is suggested that the lipogranulomatosis error of lipid metabolism. Its enzymatic basis

I

and Unman [I ] dewho died of an illness with unique clinical and pathologic features. Soon after birth tender and swollen joints and a hoarse, weak cry developed. Joint involvement became increasingly severe and generaliLet1, multiple subcutaneous and periarticular nodules appeared, and there was slight to moderate enlargement of lymph nodes. The liver was of normal size or slightly enlarged; the spleen was not enlarged. There were frequent episodes of fever and clyspnea, ztssociated with pulmonary infiltrates. Since s

1957 Farber,

scribed

three

Cohen

young

children

syndrome represents is as yet undetermined.

an

inborn

this first report, seven additional patients (including the present one) have been described (Table I and [l-7]). In five of these patients the clinical course closely resembled that described by Farber et al. [I] whereas the remaining two have had a milder form of the disease. One patient (Case IV) had severe involvement of joints, larynx and subcutaneous tissues when first observed at twenty-three months of age. A recent follow-up [6] indicates that this patient, now twelve years ot age, is without apparent neurologic deficits even though severe joint deformities and cutaneous

*From the Neurology and Pathology Services and the Joseph P. Kennedy, Jr. Memorial Laboratories at the Massachusetts General Hospital, Boston, Massachusetts 02114; and the TValter E. Fernald State School, Waverley, Massachusetts 02178. This investigation was supported in part by U.S. Public Health Service Grant NB-02672, and Grant No. 449.C5 from the National Multiple Sclerosis Society. Dr. Hubert J. Wolfe was a Fellow of the Medical Foundation. Requests for reprints should be addressed to Dr. Hugo W. Moser, Walter E. Fernald State School, M’aterly. Massachusetts 02178. Manuscript received January 10, 1969. +Present address: St. Louis Children’s Hospital, 500 South Kingshighway, St. I.ouis. Missouri 63110. $Prrsent address: Ft. Dietrich, Frederick, Maryland 21701.

870

Farber’s

Lipogranulomatosis-Moser

et al.

TABLE I REPORTED CASESOF FARBER’SLII’OGRANULOMATOSIS Case No. Sex

Reference

Age at Death (mo.)

CSF Protein (mg./lOO ml.)

Neurologic Status

.. .

Other Findings

...

I

F

Farber

et al. [I]

14

...

II

F

Farber

et al. [I]

14

42-78

III

M

Farber

et al. [I]

7

130-41s

IV

M

Zetterstriim

(Alive at 12 yr.)

Normal

.

V

M

Abul-Haj

20

... ...

“Severe retardation in mental and motor sphere”

. .

v1

M

Schanche et al. [I]

14

...

“Terminal mental deterioration”

. .

160

Areflexia

VII’ WI1

F M

[2] et al. [?I

Cogan et al. [5]

9%

Moore, R. as cited by Cracker et al. [6l

IX

M

Cracker et al. [6l

X

M

Rampini,

Clausen [7]

22

(Alive at 6 yr.) 7

Hyporeflexia

Sib of patient (Case II)

Mild macular cherry red spot

. .

...

...

...

...

Terminally areflexic Babinski sign, no pupillary reflexes

Normal

.

.

525 at 2 mo. Progressive flaccid paralysis, 130 at 2I/* mo. absent reflexes, hydrocephalus treated by ventriculoatriostomy at 3 mo.

* Present case.

granulomas have persisted. Cracker et al. [6] described a patient, now six years of age, who is entirely well except for moderate involvement of the joints and subcutaneous tissues. Clinical evaluation of nervous system function in the young patien,ts has been difficult because the deformed and painful joints have modified the infants’ responses. Three patients (Cases II, III and VI) exhibited such severe changes that there could be no doubt of nervous system involvement. One patient (Case II), who died when she was fourteen months old, showed diminishing reactions to all stimuli during the terminal stages to the extent that she did not respond to venipuncture. Her deep tendon and pupillary reflexes had disappeared, whereas a Babinski sign persisted. Another patient (Case III) showed hyporeflexia and an elevated spinal fluid protein concentration (130 and 418 mg. per 100 ml.) ; tonic neck reflexes became evident before his death at age seven months. One patient (Case v) showed “severe retardation in motor and mental spheres” [?I. Another (Case VI) died at fourteen months of age after “terminal mental and physical deterioration” [4]. One patient (Case x) showed progressive paralysis and loss

of deep tendon reflexes in the legs. In addition, this patient had communicating hydrocephalus, and ventriculoatriostomy was performed when the child was three months old; this pro cedure relieved the hydrocephalus but did not otherwise affect the progress of the disease. For neurologic abnormalities the other patients, are not mentioned (Cases I and VII) or denied (Cases IV and IX) . Histologic studies have revealed an apparent dichotomy between the lesions of joints, nodules and viscera and those of the nervous system: the former resembled those of the histiocytoses whereas the latter have resembled the lipidoses. In the joints and viscera, there was granuloma formation and a variable accumulation of foam cells distended with periodic acid-Schiff positive (PAS-positive) material. The periarticular and subcutaneous lesions, in addition, showed fibrosis. In the nervous system the main abnormality was distention of neurons with PAS-positive cytoplasmic material. The anterior horn cells and large nerve cells of the medulla, pons and cerebellum have been most involved, whereas the cerebral cortex has been least affected; the autonomic and visceral ganglia have also been AMERICAN

JOURNAL

OF

MEDICINE

Farber’s

Lipogranulomatosis-MoJer

involved. White matter has shown focal myeh degeneration and gliosis of variable severity. 1Yle cause of the lipogranulomatosis syn drome is unknown. Farber et al. [I] have emphasiyed that this syndrome forms a bridge betkveen the histiocytoses, such as eosinophilic granuloma or the Hand-Schiiller-Christian disease, and certain inborn errors of metabolism. Genetic data are insufficient to decide this point, The disease has occurred sporadically except in one family in which a brother and a sister wrre affected [I]. Although this pattern is compatible with an inborn error of metabolism transmitted by an autosomal recessive mode of inheritance, it could also occur if the disorder were due to an exogenous agent. Farber et al. in their initial report [I] proposed that fibrosis and accumulation of nonfoamy macrophages were early events, that lipidization of macrophages was a late phenomenon and granuloma formation an even later reaction. This formulation places the lipogranulomatosis syndrome among the histiocytoses but, as these investigators have emphasized, it does not provide an explanation for the neuronal storage. The nature of the PAS-positive material which accumulates within the foam cells ant1 the neurons is also unknown. Two groups of investigators [1,7] considered it to be a glycolipid; one group [3] concluded that it was a nonsulfated acid mucopolysaccharide. I‘11e (linic,al and pathologic data to be presentcd here are in close agreement with those previously described. Our biochemical and histochemical data indicate that the PASpositi\“e storage substance is a ganglioside. Evcrr more striking is a ten- to sixtyfold excess of free ccramide. Because of this, we believe thar the lipogranulomatosis syndrome represcats .~n inborn error of lipid metabolism. CASE

REPORT

This patient was admitted to the filassachusctts General Hospital (MGH No. 1322400) at four

and

:I half months of age because of painful joints and feeding difficulties. When she was IWO weeks old painful swellings of the interph:d;mgeal joints developed and caused her to swollen

hold her hands flexed; she would cry when they were extended (Fig. 1). These symptoms persistctl and by the time of her first admission it had bccome diffkult to feed her. Prt:pnancy and birth had been unex-entful; the infant’> birth weight wa\ 5 pounds and 13 ounces;

x51

et uE.

ante of han(ls at

tout

half months of age. deli\ ery was spontaneous and uticoniplicarccl. .\ six year old brother suffered from hay fc\cr and asthma, and there were five year old twitI> (;I boy and a girl) who are well. I’herc wa5 rt Ilistory of rheumatoid arthritis affecting the niotlir~r, a m;~ternal uncle and aunt and the maternal gl.,lndmother. When the mother was S~V~IILC‘C’II ye;tr, old migratory 1)olyarthritis developetl 1or whit h she required hospitalization and home irc;il~nc~~t for a fu!l year. The maternal uncle h;ls I\.~tl bwcllings at tllc proximal interphalangeal joints ~lifl((’ .~n attack of arthritis at age eightec.tl. \ p;lt”rII;ll uncle has diabetes. On physical examination of the patient, weight 99°F.. puk 1~10 per was 5.0 kg., temperature minute and blood pressure 80 mm. Hg. l’he (hiId was alert but cried when handled. Her \oicv was hoarse and barely audible. -I‘llrre TV;IS swelling of the irlterphal;lrlgeal joints of all Ilrlgvr-5 211~1 crythema of the dorsum of both great tot’> ai tlrc metacarpopl~alangeal joints. .I 11c’ li\c-r w;~s ~~tipable 2 fingcrbreadths below the riglit CO\Ia1 m.11.. gin. The spleen was not palpable. 7‘11~1c 1, .I) a

852

Farber’s Lipogranulomatosis-Moser

et al.

regular heart beat, and no murmurs were audible. The patient was given a two week therapeutic trial of salicylates, without clinical improvement. A biopsy specimen of a nodule near the right ankle showed fatty macrophages containing PASpositive material. The child was discharged from the hospital at four and three-fourths months of age. At a clinic visit at age five and a half months the child was found to have continued irritability. She was able to take only 3 ounces of milk four times daily. Weight was 5.3 kg.; height 62.5 cm. New nodules had developed on her hands and feet. She was admitted to the hospital for the second time at eight months of age. This admission was occasioned by increased difficulty in feeding. ln addition, the soft tissue swelling on the dorsum of the left foot was larger, swellings had appeared at the knees, and there was increased involvement of the wrists and fingers. The ophthalmologic consultant (Dr. David G. Cogan) found that she had normal visual function, as evidenced by her alertness and normal response to an opticokinetic drum. The corneas were clear. There was borderline pallor of the optic discs which was probably pathologic, particularly on the right side. The entire retina showed slight peppery granulations which were considered to be borderline normal. In the central areas about both maculas there were abnormal grayish opacifications, the centers of which stood out as cherry red spots [5]. The child was unable to hold her head up and she was hypotonic. Triceps, biceps and knee jerks were l+; ankle jerks and plantar responses could not be elicited. The patient’s third admission at eight three-fourths months of age was necessitated markedly increased feeding difficulties: she

and by was

able to take only 1 ounce of food per feeding and was unable to suck although she was able to take semisolid foods or food introduced with a syringe. In addition, many new subcutaneous nodules had appeared along the lumbar spine and in ail extremities. Breathing was more difficult, with retraction of the rib cage during inspiration. The eye findings were unchanged. Fluids were administered by gavage feedings during this hospitalization. The neurologic examination was performed by Dr. Philip R. Dodge and the report read as follows: “Head circumference, 40.5 cm. Wide-eyed infant, relatively alert. Full visual fields; with facial asymmetry, better closure of the left eye than the right. Hoarse cry, slight spastic catch at the ankles on passive manipulation. Deformities of the extremities mainly due to local disease. No deep tendon reflexes obtainable; plantar responses flexor; sensation and power appear intact within the limits of examination.”

Fm. 2. An early skin l&on involving the tlcrmis and subcutaneum. Note the separation of individual collagen bundles by distended cells with clear and foamy cytoplasm. Van Gieson stain, original magnification x 500. TABLE

II

RESULTS OF LABORATORY

Result

Test Hemoglobin (gm./lOO ml.) At 4M mo. of age At 8 mo. of age Hematocrit (70) White blood cell count (cells/cm mm.) DiRer.ential Urine (cells/high power field) Albumin Glucose Fasting blood sugar (mg./lOO ml.) Serum Creatinine (mg./lOO ml.) Total protein (gm./lOO ml.) Albumin (gm./lOO ml.) Globulin (gm./lOO ml.) Uric acid (mg./lOO ml.) Acid phosphatase (normal, O.Ol0.56 Sigma units) Latex fixation test Antistreptolysin titer (Todd units) < Lupus erythematosus preparations Urine and blood amino acids Berry spot test for urine polysaccharides Erythrocyte sedimentation rate (mm./hr.) Uncorrected Corrected Spinal fluid (at 8 mo. of age) White cells (per cu. mm.) Red cells (per cu. mm.) Protein (mg./lOO ml.) Sugar (mg./lOO ml.) Gold sol curve Serum iron (pg./100 ml.) Serum iron-binding capacity (pg./1OO ml.) AMERICAN

TESTS

JOURNAL

10.5 8.9

26

9,900-13,600 Normal 2 None None 83 0.6 and 0.5 7.7 4.8 2.9 7.4 and 5.2 0.92 Negative 100 Negative Normal Negative

51 12 Clear and colorless 3 10 160 47 5555544221 84 432 OF

MEDICINE

Farber’s

Lipogranulomatosis-Moser

41 riinc

monrhs of age the infant was trnnsto 111~ \Valter 11. Fernald State School. I hut she had a fever with temperature elevations to I05 I;.. increasing dyspnea and tachycartlia; tlrc li\er edge was 6 cm. below the costal Inargin. MIC was digitalixd and started on oxygen ;rntt her condirion seemed [ Irtwpy ant1 antibiotics, however, her fever autl IO impox .Z week latel, lrrl-cd

~vt’l e 1espirat.ol-y di\tres\ recurred, and she died On the last day .il ;tgc. riinc and .I ha!l months. $)I lilt, ;I pctechial rash developed.

l’os~niortem examination was perfornictl four .uJd :I half hours after death (RIGH Autopsy No. ‘l~lw Ixxly measured ti2 cm. iJI length “88%~ jnorni;il, 70 cm.) and weighed 10 pounds (norsubcutaneous rnal. I!).5 po"l1'lc) . Firm yellow-gray IIO~LI~C wire prc5ent 01 cr the wrists, metacarpopll;~l;~r~gcaljoints, interphalangeal joints (Fig. I) .1nt1 I I~oracolumbar spine. The axillary, inguinal, i,;ir:l-aortic, pel\ ic and portal hepatic lymph nodes were’ gI-eatlv cnlargcd, firm and yellow-tan. 1 lI(, li\c,r \vrcighed 230 gm. (normal, 274 p.) rlltl \l~oa-~tl IIO .Ibnormality. The spleen weighed .!I) gnu. (IIoI-nIa1. 22 gm.) and appeared normal. 7 IIt, lar~ns showed yellow-tan firm nodular deposits. 1nt71sui irlg up to 1 cm. in size, both anrcrioI to Ill<. epiglottis and in the pyrilorm \inIJs(.\. 71-lli\ Inaterial displaced the base of the <:piglot tis po5’criorly so that the glottis and esowere partially obstructed. The phageal orifice \~~a1 cortls and trachea were normal. l‘he thyroid, thymus, mediastinum, pleural and pericardium and bronchial p<*I-itoncal cavities, iyml)lL node5 showed no abnormality.

ct a!.

‘I IIc riglit luq weighed 74 gm. antl lhr lelt lung 46 gm. (IIornIal. 54 and 51 gm., respecti\ cly) . ‘Ilie lower Iol~es of both lungs, the middle lot)<. ol the rig-lit luq and the lower se,gme~Jt\ of the upper lobes 01 lioth lungs were IirJn. tlrep red an(l norIcIepitarit. -1‘lie heart wcigll~tl 30 !;ni. (norlllal, :3i q11.) ant1 was normal. k:;t( II kitlrrc~ weighed 28 gm. (normal, 31 gm.) and sllow-ctl no abnormalit!. -l‘he ptrointestinal tract, c,11do( 1i1le organs, genitalia and bone marrow wc’rc’ ~iormal. The hin 5liowetl *Jo abnormality on xi sh\ t'\.i*iiinatioii. On mici o\copic cxaniination of a +ul~ Iit;In(~oiis nodule th(, I~orInaI dermis and subcut;t~Icons tisstIe were found to he replaced by :I pl~omorphic, focally Iiecrotic iirfiltrate of foam cells. llihiioc\tc.s, lymplloc>tc,\ and plasma cells (Fig. 2 atltl 3) In the lar\iis the sol t tissue nodules were coml~~s~d of tlense collagen bundles interspersed \\,itli loam cells, histiocytes and niescncliymal tell\. IIr tlte luqg occ:hional ;(I\ coli and bronchi u rr(‘ fillrd liith I.Ir,gc loam cells. The areas ol conrr)litlation contained an admixture of foam cc.11~. lvrnl~l~oqtes, plasm.~ cells and rare giant cells. I 11~I-ed pulp of rhe spleen contained numcro~I\ Io,lm cells, arld the \shitc pulp was dcpletctl 01 Iynl)liocytvs. 1‘11crc ~~a5 nliliimal vacuoliratioIl 01 li\cLr cortl cells. In tllc lymph nodes I~UIIIC~C)II~ loam cells wcrc 1”.esent in the cortex and mc~tlulla~-v sinuses, focally disrupting the normal notI; aichitecture (Fig. 4). Foam cells were seen sc;~ttcred among t.llV JIormal 1IcIrlittopoieLic elements 01 the bone marrow. 1 leart ant1 kidneys \IIoM.c(~ no microscopic al)IIoI-malities. There was marked ncuronal loss in tlic frolltal, parietal and occipital lobe cortex, M.itlI accompanving iibro1ts gliosis and microglial proliferation (Fig. 5) : the secoIlt1 and third layers 01 cor-

Farber’s

874

cortex FIG. 5. Parietal is a marked proliferation cation X 115. FIG. 6. number.

showing severe of pleomorphic

Neurons of anterior horn Cresyl violet stain, original

FIG. 7. Prominent neurophagia original magnification X 190.

Lipogranulomatosis-Moser

nerve cell microglia

loss with distention of many of the remaining neurons. There and fibrous astrocytes. Cresyl violet stain, original magni6-

showing marked cytoplasmic magnification x 500. of

swollen

et al.

neurons

tex were most severely affected. Loss of myelin was slight and was confined to regions of neuronal depletion. The hippocampus showed mild neuronal loss and reactive gliosis. Some of the pyramidal cells of the hippocampus were swollen, with peripheral displacement of their nuclei, suggesting cytoplasmic accumulation of storage material. The neurons of the brain stem nuclei were also distended with storage material This change was more prominent in the nuclei of the tegmentum of the brain stem than in the base. The putamen and caudate nuclei showed neuronal loss and fibrous gliosis; only an occasional neuron had distended cytoplasm. The cerebellum showed a normal complement of Purkinje cells, and some of these appeared distended. The anterior horn cells of the spinal cord show,ed marked distention of nerve cell bodies with peripheral displacement of their nuclei (Fig. 6). Dying neurons were seen in these areas, and there were a number of neuronophagic nodules (Fig. 7) . The femoral nerve showed some disruption of its nerve fibers and focal accumulations of Schwann cells. Posterior root ganglion cells showed accumulation of storage material. A granuloma was found in the sternocleidomastoid muscle, which was otherwise normal. Descriptions of the eye changes iu this patient have already been published [5]. The main al,-

distention.

in an anterior

horn

Glial

of

cells

spinal

are

greatly

cord.

Cresyl

increased

violet

in

stain,

normality was the accumulation in the retinal ganglion cells of a birefringent PAS-positive, nonmetachromatic material which could be estractcd with a variety of lipid solvents. HISTOCHEMICAL

STUDIES

Methotls. For the study of lipids, fresh surgical and postmortem tissues were frozen rapidly on dry ice, cut at 10 to 20 p in a cryostat at - 18”~. and placed on slides. The sections were examined for birefringence, and the following histochemical stains were employed: periodic acid-Schiff (PAS), performic acidSchiff (PFAS) , alcian blue, phosphomolybdic acid reaction, Sudan red and Sudan black B [8] as well as the Bial reaction [9]. In addition, the PAS reaction was performed following diastase and hyaluronidase digestion. The staining reactions were also performed on fresh frozen sections which had been extracted with chloroform-methanol 2 : 1 V/V at 37’~. for twenty-four hours or wi’th distilled water at room temperature and 60”~. for one hour. For the study of tissue polysaccharides, 4 mm. thick blocks of fresh tissue were frozen rapidly in isopentane chilled with dry ice and liquid nitrogen. The blocks were then

Farber’s

Lipo~aIlulomatosis-~0~~~

et al.

8175

H

I I(.. 3. krcsh, Frozen, urrstaincd section of spinal cord siel\ctl with polarLet liglrt and showing birch-ingent material within the cytoplasm of the anterior horn I ells 0rigin;tl magnification X 120. FIG. 9. Fresh frozen section of subcutaneous notlule illustrated in Figure 3. Intense PAS-positive cl toplasmic staining of the foam and spindle cells. P1\Shfmatoxylin stain, original magnification X 225. 1;~;. 10. Fresh frozen section of the subcutaneous nodule illustrated in Figure 5 but extracted in chloroform-metharrol prior to performing PAS reaction. Note f he loss of the intensely PAS-positive cytopiasmic staining. PAS-lrc,nratox)lirr stain, original magnification K’ iiO0.

ill absolute ethyl alcohol at __70 I:. in an it-e chest for two weeks and then I~ough t to rown temperature. The tissues were r~~betltletl in a methyl methacryla~te-glycol dislearatc mixture accorcling to the method ontiined by Catf~ey [IO_i, sectioned with a steel kniIe on a rotary microtome at 2 to 4 p, floated onto albuminized slides from a nonaqueous Illetlillln and placed on a warm hot plate to tl1.y. ‘i‘he slides were then placed briefly in ;icccone, transferred to a 95 per cent ethyl alcohol solution and passed at three to five minute intervals through a series of solutions containing I per cent toluidine blue in 80 per tent, ‘70 per cent and 50 per cent ethyl alcohol, respe&vely. They were differentiated briefiy in 50 per Cent ethyl alcohol dehydrated in acetone, cleared in xylene and mounted. Other sections were stained with alcian blue and aqueous cotuidine blue, both before and after hyalurotlidase digestion [II]. sneeze-sul)stitut~~~

%OL.

4 7 ,

DECEMBER

1’369

1~csults. Similar histochemical properties were noted for the material distendii~g the neurons of fronta cortex, hippoc’anlp~t~ 2nd brain stem, the Purkinje cells and the ;Illt(*l iol horn cells. It showed strikin, u birefri ngence with crossed polaroids (Fig. 8) and ir~renscly positive I’,\S and alcian blue react ions wftich were abolished by prior extraction ot the frozen sections in chloroform-met.hanol (Fig. 9 and 10) hut were unaffected by extraction with distillctl water or digestion with diastase or hvaluronidase. The material was equally posit’ive with the phosphomolybclic acid reaction but stained only a slate grey with Sudan biack B and wx unreactive with Sudan red. There was no rnetachrornasia with cresyl triolet or toluidine blue. The Bial ant1 PFAS reactions were negative. In the visceral organs, the properties of the lipid deposits were similar in many resl)ects to those noted in the neurons. There WIS I’.\!?

Farber’s Lipogranulomatosis-Moser

876

TABLE HISTOCHEMICAL

REACTIONS

Specimen Brain Lung Liver Lymph node Spleen Subcutaneous

OF STORED

LIPID

Birefringence 4+ 4+ 4+ 4+ 4+ 4+

tissue

et al.

III

IN VISCERA

AND CENTRAL

NERVOUS

SYSTEM

PAS

Sudan Black B

Alcian Blue

4+ 1-2 + o-1+ 2-3 + I-2+ 2-3 +

1+ 3+ 34-f 3+ 3+ 3-4 +

4+ 1+ 0 l-2 +

*

Phosphomolybdic Acid 4+ 4+ 4+ 4+ 4+ 4+

o-1+ l-2 +

* Graded 0 to 4 plus

positivity of varying degree in the cytoplasm of the vacuolated cells noted in the routine histologic study of lymph nodes, spleen, lung, subcutaneous nodules and liver. This reaction was abolished by prior chloroform-methanol extraction but unaffected by distilled water. Birefringence and positive phosphomolybdate reaction was also demonstrated whereas Bial and PFAS reactions were negative. However, the visceral deposits differed from those of the central nervous system by having a more intense staining reaction with Sudan black B and a weaker affinity for alcian blue. The histochemical findings are summarized in Table III. Histochemically, no polysaccharide accumulation was demonstrable in any of the organs studied. BIOCHEMICAL

Levels

of

Major

STUDIES

Constituents.

Methods:

Pertinent tissue samples were frozen at the time of postmortem examination and were stored in sealed plastic bags at dry ice tem-

peratures. As controls (Table IV), we used tissue samples from patients who died of other diseases, matched as closely as possible for age and stored under identical conditions. In most instances, the lipids were extracted and purified by the method of Folch et al. [12]. However, for complete extraction of gangliosides this procedure was modified as described by Suzuki [I?], and N-acetylneuraminic acid (NANA) was estimated as described by Suzuki [13]. Lipid hexose was estimated by a semimicromodification of the orcinol method of Sorenson and Haugaard [If], and chloroformmethanol extractable protein was determined by the method of Lowry et al. [IS]. Individual phospholipids were isolated by the method of Rouser et al. [16], and free fatty acids were measured by the method of Schnatz [17]. To estimate liver mucopolysaccharide content, the defatted tissue was digested with papain and dialyzed [18]. The acid polysaccharides were then precipitated with cetyl ammonium bromide [19] and the glucuronic acid

TABLE CONTROL Age

Organ

Liver

at Death 2 4 7 10 18 23

mo. mo. mo. mo. mo. mo.

POSTMORTEM

TISSUES

IV

USED IN

Diagnosis Christmas disease, ? meningitis Tracheoesophageal fistula Congenital heart disease Traumatic pericarditis Anoxic encephalopathy Bilateral renal hypoplasia, ruptured appendix

STUDY

Organ Kidney

OF FARBER’S

DISEASE

Age

at Death 23 mo. 3 F

Diagnosis Head injury Microcephaly

Lung

1 day 6 yr. 11 yr.

Stillborn Cutaneous burns and infection Mongolism

Brain

6 mo.

Pulmonary atresia-ventricular septal defect Biliary atresia Neuroblastomametastases to liver but not to brain

Lymph node 2 days 7 wk. 3 r. 3 Yr* 3 yr.

36 weeks gestation- Dandy Walker malformation Coarctation of aorta postoperatively Metachromatic leukodystrophy Tay-Sachs disease Microcephaly

7 mo. lth yr.

AMERICAN

JOURNAL

OF

MEDICINE

L.i\er

106

31 * G1.G *

Gl.5

3

359

6.5

2

f .xxithint

50.7

52.8

4~)hingomyclini_

16.1

5.7

21.8

4.6

I’hosphatitl\ illohitolt

2,630

182

212

gluculonic

4,130 atid

128 *

5

LJPIDS AND POLYSACCHARLDES

3.5

3.05 2

...

..

...

...

510

1,120

19.0 55.8

6, .!, 11.1

17.7

11.1

T..5

..a

62 *

11

T30

23 & 7

57 *

6

20’,

228 _c 20

LR’ L.,‘\tP”

NOD,? AND SUlK,;~t’,NEOL,S

280

LIata “LJpper phase” [12]: Lipid NAKA

Farber Control (!)I/zmo.) (2 da>h) 210

“f.owcr phase” [[?I: Total lipids as I:;, fr<+ weight

4.98

Lipid hcxose

321

sphingosine

Polysacrharide glucuronic acid

2,320

7.3

5.75 465

Control (3 yr.) 28

7.02 219

.

.

* Expressetl as micrograms per gram of fresh weight, OII the dorsum of the right foot was performed when \\ithin a few minutes and stored in a dry ice chest. rhe spinous processes of the lower cervical vertebrae. I- Expressed as gamma per gram of dried, defatted DECEhlBER

1969

Control (3 ?I-.)

N”,,V,

7

.

lipid

NANA

C*

Farhe, C:ontrol Control Control (SJvi 1110.I (7 Irk.) (2 ).I-.) (2 Il..) 77

39

25

65

49

3.OG

i .7.5

2.39

9.50

201

2X0

123

243

296

.

5GO

.

. .

IO.62

30 c

hlrscmteric L.ymph Notles

________.-

,4 7,

0.9

. .

496

Femoral Lymph Nodes

1’01..

.

...

Pol)aaccharitle hexosamine .. . 101 & 2 44 * 2 I;rc~ fatty at id as cLM/nlg. “lo\\-erphase” lipids 0.278 * 0.036 0.838 __# Expressed as micrograms per gram of fresh weight, unless otherwise indicated. -1 Expreaacd as percentage of the total phosphorus r-etovered from the thin layer plates [Ih]. Variations shown for control values represent average deviations except for “upper phase in the li\er which represents the standard deviation.

lkramide

23.6

29.4

I seline anrl


4,330

IO.3 _ I

33.9

X.8

0.14

1.88 *

4.19

810 -+ 50 11.4

I c*thanolamine+

f’hosphatitl\

..

..

148 -I_ 40 2,113

Iti.3 k

8”

3,230 I+ 700

1,475

Farber

.

3.RG _c 0.34

G.93 6,290

Cont1ol

_e 0.11

i.ti5

8G

Kitlne,

L,ung

~Farl,cr

Colltl.ol

E‘arbc1

1,X5+

. .._

Iliops\ \utops) 410

1i0

1Xl 3,100

1,450/-

unless otherwise indicated. Biopsy of a subcutaneous nodule the patient was nine months old. The material was frozen The nodule obtained at the time of autopsy was located over tissue residue.

Farber’s

878

et al.

Lipogranulomatosis-Moser TABLE VII NERVOUS

Cerebral

“Upper phase” [12]: Lipid NANA Hematoside NANA as y0 total NANA Hexose “Lower phase [12]: Total lipids as% fresh weight Lipid phosphorus Lipid hexose Cholesterol Ceramide sphingosine as micrograms

White Matter (Centrum Semiovale)

570

.. .

,..

...

426

501

444

382

4.8 740

...

.. .

.. .

.. .

...

. .

.

4.74 1,205 218 10,500 622

4.47 1,180 166 9,050 468

...

...

4.98 1,403 170 9,720 309

4.82 1,350 197 8,960 525

7.80 1,575 1,750 18,900 1,773

per gram of fresh weight, except

TABLE OF VARIOUS

CARBOHYDRATE-CONTAINING CORRELATION

WITH

Degree of infiltration with PAS-positive foam cells

0

Ganglioside

1.25 ...

Polysaccharide glucuronic acid

15.2 2,820 5,280 34,700 642

...

...

...

.

230

:::

9.39

9.82

... ...

. . .

.. .

..

11.1

. ...

indicated.

VIII

HISTOLOGIC

Mesenteric Lymph Node

“Lower phase” hexose

8,26 1,820 2,310 18,000 384

where otherwise

SUBSTANCES

Kidney

NANA

5.22 1,275 550 10,400 452

210

250

we did not estimate the free ceramide levels in control lymph nodes. There was a three- to fivefold excess of free ceramide in brain white matter, whereas essentially no excess was demonstrated in grey matter. The other rather consistent abnormality was a moderate elevation of lipid NANA, which correlated roughly with the degree of foam cell infiltration (Table VIII) . Total ganglioside levels in the brain were not abnormal. “Lower phase” glycolipids, which include all carbohydrate-containing lipids devoid of

was measured [20]. For tissues such as lymph node, when only small quantities were available, the precipitation with cetyl ammonium bromide was omitted. Results. As shown in Tables v, VI and VII, the most striking abnormality was an excess of free ceramide in the visceral organs: in the liver it was 60 times that in the control, in the lung 30 times and in kidney 10 times. In Farber’s disease, ceramide levels in the lymph node and nodule were comparable to those in the liver. Due to the small samples available,

Data

Spinal Cord (Midthoracic Segments)

Farber Control Control Control Farber Control Control (9/12mo.) (6mo.) (7mo.) (lr/ayr.) (91/smo.) (7 wk.) (3 yr.)

content

EXCESS

LIPIDS+

Cortex

Farber Control Control Control (9Gmo.) (6mo.) (7mo.) (ll/,yr.)

Data

*Expressed

SYSTEM

IN FARBER’S

LIPOGRANULOMATOSIS;

FEATURES

Femoral Subcutaneous Lymph Node Nodule

Liver

Lung

1+

1+

2+

3+

3+

1.7

2.8

5.1

8.4

...

1.3

2.4

0.86

1.1

1.1

2.2

0.90

...

... ...

The values shown represent the ratios between levels of tissue constituents in Farber’s disease and the NANA excess controls. The ratios were computed from the data in Tables v and VI. Only the ganglioside correlates with the degree of foam cell infiltration. For the subcutaneous nodule, these ratios cannot be computed, as there were no comparable lesions in the controls; however, lipid NANA concentration in the Farber’s disease nodule was comparable to that in the femoral lymph node.

NOTE:

AMERICAN

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OF

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Farber’s

Lipogranulomatosis

ABCDEFGHI

-. Moser et al.

A

BCDEF

GH

I

KI(.. I I. I.(,//, thin layer chromatography plateof liver glycolipid fractions. The total lipid extracts hat1 hccn ~~lv)~l)etl on Vloxisi! columns, which were eluted with chloroforms and then with chloroform-mcthallol 2:l cl’/\‘) ;\liquots ot the .econtl cluate were spotted on a thin layer plate, tlrwloped with the solxc~nt qstem N plol)~~~~ol:;ln~n~c,ni;~:\\.3t(fl c12.I : :!) \‘;\‘:‘V ;tlltl splayed with an y-llapthol-sulfuric acid srrlulion I_lVi; untler these conditions glycolipitls \how up as l)urplc-pink spot\, rvhereas the other lipick tlc~eiol~ a brown color clue to challillg. .2, R an(l (‘. c011llol li\cr of liver of patient with Fal her’s tliwaac. ecltti\a ,xtl;icIb eclui\alellt to 5 01 G mg. of total lipid. I), extract standard. F, ceramitle tlihexosidc. G, ceramitlr tlihexoGtle. t I, ient to i.7 mg. of total lipid. E. glohositle
Ileuraminic acid, were elevated in the liver but did not di&r- from control levels in the lung or lymph node or brain. Their composition was studied qualitatively by means 01 thin layer chromatography of fractions which had previously been partitioned on Florisil@ coltmina. In liver, lymph node and nodule, the pattern appeared similar to that of the conIrols; in brain grey matter, there was a slight (zscesh of a component with a mobility similar 10 that of dihexoside (Fig. 11) . Rampini and Clausen [7] report the presence in Farber’s disease of an unusual glycolipid containing ceramide, glucose and galactosamine, but free of galactose. It is likely that such a compound would be found in the Florisil fraction shown in Figure 11, and our failure to observe an unusual spot suggests that this substance M‘;IS not present in our patient, at least not in large quantity. Polysaccharide levels were increased in the liver but not in the lung or lymph node. Total phospholipid levels were normal in the brain and liver but moderately increased in \nL.

.47,

DECEMBER

1969

the lung. I:r;itiionation of the pliospholipids in liver and lung revealed a normal percentage of lecithin and sphingomyelin. There was a moderate increase of phosphatidylserine and inositol phosphatide, but our method did not allow us to determine if the increase applied to one or both of these constituents. Finally, in both liver and lung there was a diminished percentage of phosphatidylethanolamine. This may have resulted, at least in palt, lrom postmortem autolysis. The fact that free fatty acid levels in the liver were increased suggests that a moderate degree of postmortem autolysis had taken place in that organ. Isolation of Ceramide and Determination of its structure. Ceramide was separated from the other sphingolipids by chromatography on Florisil columns using 40 cc. of solvent per gram of Florisil. In the elution scheme: hexane, hexane:diethyl ether 55 : 25 (V,V) . hexane, chloroform, chloroform : methanol 19: 1 (V/V), chloroform:methanol 2: 1 (V/V), the compound was contained entirely in the

880

Farber’s

Lipogranulomatosis-Moser

et al.

FIG. 12. Thin layer chromatogram of Farber’s disease ccramide, isolated from liver (A), authentic ceramide (B) . Solvent system chloroform:methanol:glacial acetic acid (192:5:8) (V/V/V) Sulfuric acid-dichromate spray. Spot 4 represents ceramide containing hydroxy fatty acids, spots 2 and 3 ceramide containing normal fatty acids; spot 1 is unidentified, it does not contain sphingosine. FIG. 13. Demonstration by thin layer chromatography of ceramide accumulation in the liver of the patient with Farber’s lipogranulomatosis. Solvent system chloroform: methanol 95:5 (V/V) stained with sulfuric acid-dichromate spray. A and B, extracts from liver of patient with Farber’s lipogranulomatosis. D, E and F, liver extracts from control liver samples. C, authentic ceramide.

chloroform:methanol 19: 1 fraction. With the exception of the diethyl ether, all solvents were redistilled prior to use. Three solvent systems were used for chromatography of this ceramide fraction on 0.5 mm. I, chloroform:methanol: silica gel G plates: glacial acetic acid 192:5:8 (V/V/V) (Fig. 12) ; II, chloroform:methanol 95:5 (V/V) (Fig.13) ; III, chloroform:methanol:water 92:28:3 (V/ V/V) . In system I, four major components were found: (1) Rf 0.63, (2) Rf 0.39, (3) Rf 0.33 and (4) R, 0.13 (Fig. 12) . The Rf values of components 2 and 3 were identical to those of authentic ceramide in the three solvent sysThe infrared spectra of compotems used. nents 2 and 3 were similar to those obtained

for authentic ceramide and agreed with the spectra previously published for this compound (Fig. 14) [21]. In order to determine their constituents, the four components were eluted individually from preparative thin layer plates; authentic ceramide was treated in the same manner. The material was then hydrolyzed by the method of Gaver and Sweeley [22], the fatty acids were extracted in hexane, and the sphingosine content of the residue was determined by the method of Lauter and Trams [23]. Components 2 and 3 contained 89.9 per cent of the sphingosine found in this fraction; component 4 contained 10.1 per cent. The sphingosine moiety was studied further AMERICAN

JOURNAL

OF

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Farber’s

L,il~~1~l‘;~rl~~lom;lto~is-i~rlose, ct ~1.

HHI

%j#_jJf_~; j//c _““-_; GUY :$ .o FIG. 14. Infrared spectrum of fret ceramide in Farber‘s ceramidc. R, componcnr 2 of compound A (Fig. 12). compound A (Fig. 12)

by gas liquid chromatography [Z?]. In liver specimens from patients with Farber’s disease and the controls, this was found to be almost exclusively C-18 sphingosine; a small amount of dihydro C-18 sphingosine and no C-20 sphingosine was detected. In the liver of patients with Farber’s disease, the fatty acids from the eluted spots were studied by thin layer chromatography and by gas liquid chromatography. We did not ap ply these technics to the free ceramide in the control tissues because of the small amount of material available. For the thin layer chromatography studies we used the solvent system described by Eng et al. [24]. The methyl esters of falty acids derived from components 2 and 3 had the same Rf values as those of authenric nonhydroxylated fatty acid whereas those derived from component 4 corresponded to those of hydroxylated fatty acids. Gas liquid chromatography of the fatty acids was perVOL.

47,

DECEMBER

1969

disease A, authentic C. component 3 of

formed with XI F-h1 Model Biometl 400 equipped with a flame ionization detector. The methyl esters of the normal fatty acids and the trimethyl chlorosilane derivatives of the hydroxy fatty acids were separated on 7 per cent DEGS and 2.5 SE-30 columns. Peaks were identified and quantified by comparison with the National Heart Institute Fatty Acid Standards [2j]. The fatty acid compositions are shown in Table IX. The nonhydroxy fatty acids contain somewhat more stearic (18-O) and oleic (18-1) acid and less lignoceric acid (24-O) than the other visceral sphingolipids for which fatty acid compositions are available. These include spleen ceramide 1261, plasma sphingomyelin [27], cytolipin [%!?I and kidney sulfatide [29]. However, analysis of the reported data suggests that these patterns vary considerably, and the result for ceramide in parber’s disease does not seem to be outside the range reported. In Farber’s disease the free

882

Farber’s

et al.

Lipogranulomatosis-Moser TABLE IX

FATTY

ACID COMPOSITION FARBER’S

DISEASE

OF FREE CERAMIDE COMPARED

Nonbydroxy Fatty Acid

Farber’s Liver Ceramide

14:o

I..

15:G

.,.

1G:O

21

Spleen Ceramide [26]

0.2 . .

Fatty

WITH

ISOLATED

PREVIOUSLY

FROM

LIVER

REPORTED

Acids

OF PATIENT

WITH

SPHINCOLIPIDS’

Hydroxy

Spleen Cytolipin H 1281

Plasma Sphingomyelin [27]

Farber’s Liver Ceramide

Fatty

Acids

Kidney Sulfatide [29]?

0.5

0.7

...

0.2

3.2

..

30.9

1 l-25

41.7

...

... ...

...

13.5

. ... ...

5.1

17:o

...

... ...

0.6

‘..

18:O

11.0

3.4

l-5

9.4

11.8

18

...

...

.

...

...

16:l

...

9.5$

IS:1

15.5

...

...

0.7

...

19:o

..*

*. *

...

0.3

.I.

8

... ...

2O:O

6.2

5.3

0.5

3.9

8.2

2O:l

...

...

0.2

...

21:o

...

...

... ...

0.3

..

..I

22:O

13.0

20.7

5-13

12.1

20.7

13

221

...

..,

...

0.4

...

11

23:0

3.8

4.2

...

5.1

10.1

11

14

...

...

*..

0.5

...

...

12.0

35.1

44-65

8.9

27.6

24:l

...

...

...

14.1

...

24 ...

24:2

*. 1 ...

. 3. ...

.., ...

0.7

...

...

0.4

...

...

... ...

23:l

24:o

25:o 2G:0

4.4

tr.

*..

...

*..

26:I

3.9

...

...

..*

.. .

* Expressedas percentage of total nonhydroxy or hydroxy fatty acids. + The original data summarized in Figure 3 of [29] were supplied by Dr. M. J. Malone. = This peak was eluted between the C18:O and C18:l fatty acids. It has not yet been identified.

ceramide contained 10 per cent hydroxylated fatty acids. Hydroxy fatty acids were not found in spleen ceramide, plasma sphingomyelin or cytolipin and in general are thought to occur mainly in the nervous system sphingolipids. However, Kishimoto and Radin [30] did demonstrate them in the visceral organs of the rat, and Malone and Stoffyn [B] reported that more than half of kidney sulfatides contained hydroxy fatty acids. In Farber’s disease the hydroxy fatty acids of ceramide resemble8 those of kidney sulfatide in that those with 22 and 24 carbons predominate. In conclusion, our results suggest that the free ceramide accumulated in the liver of patients with Farber’s disease has a normal structure. This is based upon the observation that it has the same chromatographic properties, infrared spectrum and sphingosine base as authentic ceramide or

that isolated from the control subjects. Data with respect to its fatty acid composition are incomplete since we did not analyze the fatty acids of the free ceramide in our controls. However, comparison with results in the literature suggests that the fatty acid composition of the ceramide in Farber’s disease is not abnormal. Isolation a.nd Zdentification of the Major Tissrte Ganglioside. Gangliosides were ex-

tracted by the method of Suzuki 1131 and then separated on thin-layer chromatography plates in the solvent system N-propanol-water (7:3 V/V). The major component in the visceral organs had a mobility similar to that described for hematoside [3l]. In order to quantitate this component we separated it on thin layer plates from a11 other gangliosides with the solvent system N-butanol : acetic acid : water AMERICAN

JOURNAL

OF

MEDICINE

Farber’s

Lipogranulomatosis

illoxr

et al.

X83

Thin layer chromatogram of “upper FIG. 15. phase” lipids extracted from tissue sarnplef of the patient with Farber’s lipogranufomatosis, a patient with Tay-Sachs disease. and two controls. Solvent: N butanol:acetic acid: water (3:l:l). Stained with resorcinol spray If/]. A, grey matter, Farber’s disease. B, tontrol grey matter. C, grey matter, TaySachs disease. D, control liver, equivalent to 300 mg. fresh weight. E, lymph node, Farber’s disease. F, liver, Farber’s disease, equivalent to 60 mg. fresh weight. C, subcutaneaus nodule, Farber’s disease. All the tissues shown of the patient with Farber’s disease appeared IO contain the same gangliositle (later- shown to be hematoside). It was clenrly separated from Tay-Sachs ganglioside (C) and, in the brain, also appeared to be present in greater concentration than in the control (Bj. Normal liver also contained this gangliositltr in lower concentration.

A‘

B

C

D

E

(r~(l:llO:Zl) \’ lri V) and measured its NANA content xs tlescribecl by Suzuki [13]. The componcnt with rite same mobility as hematoside ~,IS founrl to acco~mt for 61.5 per cent. of the g;lllglioside N.iNA in the patient’s liver and Cil.(i -I: 1i.5 per cent of the ganglioside NANA in ftve normal livers. Thus, although total libt’r ganglioside levels were elevated, the ganglioside pattern was normal. Inspection of thin layer piates prepared from the lung, lymph nodes and nodule gangliosides indicated tllnt this same substance was the major component (Fig. 15) but we did not quantitatcb the ganglioside pattern of these organs. In cerebral ,gray matter, white matter and spinal cord, total lipid NANA levels were normal (Table vn) . However, in the gray matter there was a significant increase in the proportion ol’ a compor~ent with a mobility similar to that of hematoside: it accounted for 5 per cent of the total brain gangliosides, which was five times that in the control and higher than that reportetl 1,) Suzuki [??‘I in norma human brain. In order to study the composition of this particular ganglioside we isolated it in larger quantities from the patient’s liver. We first separated it on preparative thin layer plates VOl,.

47,

DECEhIRER

1969

rvith the solvent system N-butanol:arctic acid: water (60:20:20 V/V/V) and then on a similar plate developed in N-propaIlol:~~ater 7:s (\‘,‘V) . The major ganglioside component was eluted from the silica gel in methanol:water -1: 1 and its molar composition determined. NANA was estimated in aliquots prior to hydrolysis. The sample was hydrolyzed with a mixture of methanol, hydrochloric acid and water [_‘_‘Ifor eighteen hours at %*c. The hydrochloric acid was evaporated in the presence of toluene:absolute ethanol 50:50 (V/V) , and the residue was taken up in either chloroform or methanol:water 4:l (V/V). Glucose was estimated by the enzymatic method of Slein [33]. Sphingosine was estimated by the method of Lauter and Trams [23]. The molar ratios of glucose, galactose and the amine sugars were determined by gas chromato~aphic technics [%I, and the substance was found to contain sphingosine-glucose-galactose-NANA in the ratio 1.05-1.00-1.15-0.91. No amine sugars were detected. Thus, both its cI~omato~aphic properties, and the nature and proportion of its constituents, suggest that this ganglioside is hematoside.

Farber’s Lipogranulomatosis-Maser

884

TABLE LESIONS

RESULTING

Days After injection

Data Ceramide (isolated from liver of lipogranulomatosis patient)

FROM

1 2

-t-+ 0 0 0 0

; 9 21

0

Ceramide (Applied Science)

0 0 0

0 0 0 0

1 5 6 7 8 9 21

Galactocerebroside

Sulfatide

x

SUBCUTANEOUS

Acute rnflammation

et al.

LIPID

Chronic Inflammation

INJECTIONS

Foam Cells

-t--t -IO 0 0 0

-t--i++-l+ 0 +++ -t--i-

0

0

0 0

0

:

0

+” 0 +++ ++++

0

+++ ++ 0

+$+ 0 +4t

+” B

z;-” +: +++ -f-Jr

+E0 ++ ++ +++ +++

I

0

++-I-

0 0 0 0

0 0 0 0

Giant Cells

0

0

+” 4-J 0 +”

++

Granuloma

+-t-i-

0 0

1 1:0

++” ++: +i; 0

1

0

1 5 9 21

0 0 0 0

Sphingosine

1 8

0 0

+0

Infant ganglioside

1

0

0

0

0

0

Silica gel

1

++

0

0

0

Sphingomyelin

++

and Roscoe Brady. We submitted liver samples to them from our patient with Iipogranulomatosis as well as postmortem liver samples from four control patients whose ages varied between four months and five years. Ceramidase activity, measured by the method of Gatt [35], was found to be normal: 0.84 m@M per mg. of protein per hour in the liver of the patient with Farber’s disease, and between 0.64 and 0.96 mPM per mg. of protein per hour in the control sampIes. Sphingomyelinase, glucocerebrosidase and galactocerebrosidase activity were also found to be normal. EXPERIMENTAL

GRANULOMAS

IN

THE

RAT

The purpose of these studies was to determine if lesions resembling the subcutaneous nodules in the lip~anulomatosis syndrome could be produced by the injection of specific

+++

+++

-t” 0 0

+++-t +++i-

0 0 0 0

0 0

0 0

++ +-t-

lipids. We used a technic described by Austin and Lehfeldt [?6]. For the injection, we used disposable 2 cm. No. 21 gauge needles equipped with a stylette. The bore of the needle was loaded from the top with the dry lipid, which was then tamped down with the stylette. This process was repeated until the needle contained a cast of lipid approximately 2 mm. long and which weighed between 0.5 and 1 mg. After the bore of the needle had been loaded, the stylette was replaced loosely so that its tip was in contact with the upper margin of the lipid. The lipids were then injected into 100 gm. Sprague Dawley rats. The injection sites were depilated with a commercial preparation. The animals were lightly anesthetized with ether, the skin was pierced with the needle containing the lipid and advanced approximately 1 cm. subcutaneously, AMERICAN

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OF

MEDICINE

Farber’s ;IIIII tlw liljitl \v;I~ tlischarged \t\ Bert?

((I 1l1:~

tip

4t.lllr (‘4. (I depit irr

\kin,

\loq

1’~ tions.

ant1

\a(‘~i II{ etl Ironi

one

jet Iion.

Iii nineteen

Ilotllllc.

;I

IK itlentifictl ,uitl

\\.c injectetl mitlc

the

iso!ated

tollowing

from

the

lif~o~r~~il~ilom;itosis. con( entrated (1~ tllin

Iaycl

The

then

(I)

material

(.\pl)lietl r;~l)hy tem

\’ V])

preparative

using

(on

the

tlesc-ribed

cernnlitle

by

contained

IV~W~C;I\ that coruaine(l gxlac

d\)

Eng

normal

tocwchwsitle

(;I gift

: (-1) gan$iosidc,

2(ribctl

tram

lipos-ran

ulomatosis

normal

infa1lt:

1’101 n

commercial

myclin,

[24],

the

comlnercial

hydroxy the of of

(5)

acids, liver

acids) as

the

(3)

;

Koscoe already

deof a

obtained

including

phosphatidyl

with

samples

lipids

sphingo-

serine,

inositol

monoglyceritles,

triglycerides,

sphingosine

‘I‘hese

actioii. bling

those

btudies in

slio\\- that

the

lesions

li~~ogranulonlatosi~

1esc’ms>;n-

drorne ran be produced by suhc~ltmeou~ Injection of certain lipids. Although the rcyonse is not specific, the ceramide isolated lrom the liver 01 the patient with Farber’\ disease IZ’;IS among thaw lvhich producetl ;I striking response.

Bra-

patient

brain

Iysolecithin,

glvt critics.

fatty

Dr.

other

sys-

patient’s

fatty

sources

hulfatitle,

phopharitlc,

solvent

or from

and

chromatog-

the

isolated li\,er

the

ceramide

layer

from

mainly

acid

with

mostly

irolnted

solvent

acetic

thin

tleveloped

with been

a5 already

by

; (2) commercial

Science)

plates,

cera-

hacl

column,

purified

cllromatography

[I’

either

isopentane.

5) jt(Lm cldorol’orm-methanol:glacial l!Q:5:8

no~lulc

and

of a patient

Florisil

ii

anal

a

could

lipids:

liver

(This

on

ribed,

in-

instances

site.

in cold

of

were

day5 after

wa5 excised

or frorcn

part

diameter,

injection

area

fisetl

of hour in-

ok twenty-two in

the

animals

to twenty-one

at the

sui-rounding

fornialin

a total The

1

under

side of the lower

millimeter5

few

the

approximately

tllorax.

385

et al.

In nlmst ill-

be seen

received

citha-

011

,~bclo~nen

rfl~

(ould

arlimals

one

needle.

lipid,

of

diameter,

mm

by arl\,ancing

the

of

Lipogranulomatosis--Mosel-

Clinical

clnd Pmlhologir

lipogranulomatosis order

of infants

or

hoai-bent3

childreu

and

subcutaneous

and,

in most

Farber’s

progr(~~si\ c: tlis-

young

i/et1 by periarticular

lonix,

I’ctrtrirc5.

is a unique

(Ilaracter~1 anu-

inataric c‘s, lower

di-

a11tl cho-

lr&rol. .l’lle to

mo5t

the

l’rom

striking

in jectetl

the patient

faricle

(Table

leuhoq ccl1

(Fig.

but

15)

also

Sphingomyelin

dltcetl

foam

cctls. 01

the

chronic

but

other

lipids

inflammation

di.

produced DECEMBFR

and

sul-

there

was

no

later.

Com-

as that

produced

the

patient’s

sphingosine after

proor giant

injection

produced

but

giant

reaction,

granulomas

hours had

and

and

a tissue from

and

By twent!;-four

Gangliositles

YoI.

cells

16)

occurred evoked

prepared

liver.

days

lymphocytes (Fig.

as pronounced

reramitle

ceramide

of polymorphonuclear

formation

cceramide

the

in response

disease

two

Granulorna

it was not

b\

IVithin

macrophages,

cells.

melcial

were

Farber’s

infiltrate

tcs,

foml

with x)

a pleomorphic

reactions

galactocerebroside,

most

acute

and

few or no foam

cells.

no detectable 1969

tissue

re-

l--KG. 17. Granulomatous response to ceramidc twenty days after subcutaneous injection. Much of the lipid remains at 1he injection site and is represented by the clear areas surrounded bv histioq tc\ and nrultinucleatcd giant tells. Hematoxylin and rosin stain, original magnification X 350.

886

Farber’s Lipograuulomatosis-Moser

motor neuron involvement in the central nervous system. The clinical and pathologic features have been recently and authoritatively reviewed [6]. Our case corresponds closely to those described previously, with the following additional features: (1) There was ocular involvement, consisting clinically of a parafoveal opacity of the retina with a mild cherry red spot and pathologically of the presence of birefringent glycolipid in the retinal ganglion cells [5]. (2) In addition to the previously described lipid accumulation in the anterior horn cells, our patient showed marked neu. ronal loss in frontal, parietal and occipital lobe cortex with cytoplasmic storage in the remaining nerve cells. (3) There was a strong family history of rheumatoid arthritis. Nature of the PAS-Positive Storage SubMost workers agree that, in Farber’s stance. lipogranulomatosis, there is storage of a PASpositive lipid. Abul-Haj et al. [j] considered this material to be a polysaccharide. However, they worked with formalin-fixed tissue which had already been treated with lipid solvents and paraffin embedded so that these lipids could have escaped detection. In our case the foam cells contained material which was birefringent, PAS- and alcian blue-positive, and these staining reactions were abolished or greatly reduced by prior extraction with a variety of lipid solvents. These histochemical properties suggested that the accumulated substance was an acidic glycolipid. Acidic glycolipids include two main classes of compounds: the sulfatides and the gangliosides. The absence of metachromatic reaction with toluidine blue argues against sulfatide accumulation such as that found in metachromatic leukodystrophy [37]. The Bial reaction for NANA, a component of the gangliosides, was also negative. In our experience, however, this does not exclude an excess of NANA-containing lipids, as the histochemical application of this reaction may produce erratic results. Thus, we found this reaction to be negative in brain sections of a patient with Tay-Sachs disease in which the accumulation of a specific monosialoganglioside has been established. In conclusion, the histochemical evidence suggests that the PAS-positive material is an acidic glycolipid other than sulfatide; its structure cannot be established by histochemical technics alone.

et al.

The most reliable manner in which to identify the nature of the PAS-positive material would be to isolate the foam cells and then to determine their composition. This type of isolation has not yet been achieved, so chemical identification has had to depend upon the less satisfactory technic of correlating biochemical and histologic data from whole tissues. Table VIII shows that in the nodule, lymph node and viscera the amount of PASpositive material observed histologically correlated with the degree of ganglioside, whereas no such correlation could be established with respect to the “lower phase” glycolipids, which include cerebrosides, sulfatides and other glycolipids which are free of NANA, or with respect to polysaccharides. It is suggested, therefore, that the PAS-positive material consists of gangliosides; as noted, this conclusion is compatible with the histochemical findings. In the case of the liver, it has been shown that these gangliosides contain mainly the “visceral ganglioside” hematoside which contains ceramide, glucose, galactose and NANA in equimolar proportions, and chromatographic studies suggest that this is also the case in the other tissues. In the nervous system in our case, the total ganglioside level was not increased. However, the total number of neurons was considerably reduced, and hematoside accounted for an unusually large proportion of total gangliosides. Thus, the quantity of hematoside per neuron very likely was significantly increased, perhaps enough to account for the neuronal distention. Free Ceramide Accumulation. The initial phases of our biochemical studies had centered on the glycolipids, since histochemical and previous biochemical results had implicated these substances. It was a surprise, therefore, when it was found that the most striking and consistent biochemical abnormality in lipogranulomatosis is the accumulation of free ceramide. In all the visceral organs that we have studied the increase of ceramide concentration (10 to 60 times control) was of the order of magnitude seen in “storage diseases.” The ceramide excess escaped detection histochemically, probably because this molecule lacks groups with specific staining reactions. In the brain, the extent of the increase was much less, 2 to 5 times control in white matter, and within normal limits in the gray matter. Study AMERICAN

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products huggested that the accumuhretl ceramirle has ;I normal structure. I\:(, had been concerned about the possibilIIY that the lrre ceramidc excess represents an How:trt< t, due to postmortem autolysis. eve,-, our control material included postmortem t i\aue samples from nineteen patients comp;11~;~1~lcto our patient with Farber’s disease IvilIl resprct to age, conditions of autopsy and tissLlr>storage. In not one of the control tissues \’surgical biopsy 01 the patient with Farber’s ~l&a~(~. This biopsy sample had been placed III ;II~ iced container immediately after reant1 thirty minutes later was trans111o~‘;l I, l‘can-ctl to ;I plastic bag which was sealed under nitrog;en and kept at dry ice temperature until It was subjected to chemical analysis. We have (oncluded. therefore, that the ceramide ,rc.curnulation in Farber’s disease is not an artet‘atl. It will be of crucial importance to determine if a similar accumulation of free ceramitlc is present in other patients with the lil,og~;lnulomato~ij syndrome. Because of the great excess of free ceramide in the subcutaneous noclule, it sh~~ultl be possible to measure this substance in ;I small biopsy specimen \qeighing from 100 to 300 mg. Further study of! pl;lsma and red cell glycolipids should also I)e of interest. C;lwolipids Other than Gangliosides and Pol~~~~accha~ides. Farber et al. in their initial report [I] noted unusually high levels of lipid !lexohe in the fresh frozen tissues of two of Iheir patients (Cases 4 and 3)) and they isolated a lipo-glyco-protein complex. As they did not define this substance completely, it is not i)os5ible to determine in retrospect if it repre\entetl ;I neutral or an acidic glycolipid or if it t ontained NAN,%. Rarnpini and Clausen, in their recent clinicopathologic report of a patient with Farber’s clisease [7], mention briefly that they isolated an unusual glycolipid, a substance containing reramide, glucose and galactosamine but neitfler NAN.4 nor galactose. Their initial report tloes not describe the chemical or chromatographic properties of this substance, and we r’annot state with certainty whether or not it ,)t ~~+d)sis

V”,

47,

DECEMBER

1969

et al.

887

was also present in the tissues of our patient. In the lipid partition procedure clescribed by Folch [/2] most glycolipitls which do not contain NANA are found in the “lower phase,” and presumably this would also be true of ttke substance mentionctl by Rampini nntl (:larl\eri. In our case, total “lower phase” glycolipid level5 were either normal or moderatel) increased (Tables v through VII), ant1 chrornatographic study of these glycolipicls tlitl not reveal any unusual compounds (Fig. 11) . These results suggest that the tissues of 0111‘ patient did not contain large quantities of this substance. 4lthough our own clata provide no clear evidence for an abnormality of glycolipids other than gangliosides, Rampini and Clausen’s interesting finding indicates that this question is still unresolved. Results with respect to tissue polys~charicle levels are somewhat conflicting. Rampini and Clausen reported increased polysaccharide levels in brain and kidney but not in liver or spleen [7]. We have found a moderate clewtion of polysaccharides in the liver. but their concentration in lymph node and lung was normal, and in Zetterstriim’s case polysaccharide hexosamine levels were normal in a nodule biopsy specimen [?]. Urinary polysaccharicle levels have been normal in tfie three cases in which they have been tested. Tlrcse data refer only to total polysaccharidc concentration, and the presence of an abnormal polysaccharide pattern has not been ruled out. However, the limited information available provides no evidence for a primary disorder of polysacchnride metabolism.

Pathogenesis of the Lipogmtllllornatosis Syndrome. Farber, Cohen and I’~man in tlleir initial report emphasized that the lipogranulomatosis svndrome formed a bridge I)et\v’ern two otherwise distinct groups of disorders. The includes Han&Schiillcrfirst
888

Farber’s

Lipogranulomatosls-Moser

The etiology of the first Tay-Sachs disease. group of disorders remains obscure, but much has been learned in recent years about the disorders of lipid metabolism. In many instances the exact structure of the storage substance has been defined, and the nature of the biochemical defect, almost always a deficiency of specific degradative enzymes, has been determined [jS]. Our data suggest that it is not necessary to postulate separate pathogenetic mechanisms for the visceral and nervous system manifestations of the lipogranulomatosis syndrome. We base this upon the observations that (1) free ceramide levels were markedly increased, even in tissues in which there was no granuloma formation, indicating that granuloma formation is not a prerequisite or precursor of ceramide excess; (2) subcutaneous injections of ceramide produced granulomas in rats. It is proposed that the lipogranulomatosis syndrome represents an inborn error of lipid metabolism. However, such a designation must remain tentative until an enzymatic defect is established. The marked excess of free ceramide in lymph node, nodule and the visceral organs suggests that the main biochemical defect in some way involves the metabolism of this substance. In contrast to what was found in the viscera, free ceramide levels in the nervous system were The normal or only moderately increased. reason for this discrepancy is not known; it may be that, as sphingolipid synthesis in the maturing nervous system proceeds very rapwhich might otherwise idly, free ceramide, have accumulated, is utilized for the synthesis of the complex sphingolipids. The mechanism responsible for the free ceramide accumulation remains a mystery. We have considered four possibilities: (1) The accumulated ceramide has an abnormal or unusual structure. However, our studies of the products of hydrolysis of liver ceramide reveal no evidence of this. (2) There is a block in the synthesis of one or more of the complex sphingolipids. In this case, the free ceramide might accumulate because it is not incorporated into the more complex sphingolipids. However, since the concentrations of sphingomyelin, cerebrosides and gangliosides. were found to be either normal or increased, there is, no evidence for deficient synthesis of sphingolipids.

et al.

(3) There is overproduction of ceramide and other sphingolipids. It should be possible to examine this possibxility by incubating a biopsy specimen or white blood cells of a patient with Farber’s disease in the presence of radioactive precursors and measuring the incorporation of radioactivity into various sphingolipids. This approach has recently been used to study sphingolipid metabolism in the white cells of patients with Chediak-Higashi syndrome; here the rates of sphingolipid synthesis and degradation were both found to be increased [39]. (4) There is deficient capacity to degrade ceramide. Our analytical data have made this an attractive hypothesis: it would account for the excess of free ceramide, as well as for smaller and variable increases in the levels of substances such as gangliosides and other sphingolipids which are one or more metabolic steps removed from the deficient reaction. However, the ceramidase activity was found to be normal. Although this finding argues strongly against the deficiency of a degradative enzyme, one cannot rule out the existence of other as yet undescribed ceramidases which may not have been included in this enzymatic assay. ADDENDUM

Since submission of this manuscript we have become aware of an additional report on Farber’s disease in which Bierman et al. [12] describe the clinical and biochemical findings in a boy with characteristic clinical features, who died when he was fourteen months old. The roentgenologic features of this case had previously been described by Schanche et al. [4] and this patient represents Case VI in our Table I. In this case polysaccharide levels were markedly elevated in the urine, and probably also in the spleen, liver and lung; the polysaccharide appeared to consist mainly of chondroitin sulfate B. No well developed granulomas were observed, histochemical study revealed no lipid excess, and there was no storage of material in neuronal elements. The results in the case of Bierman et al. thus differ markedly from those in our case: In our patient there was only a moderate excess of polysaccharide in liver, whereas total polysaccharide levels in urine, lung and lymph node were not increased. In contrast, the case of Bierman et al. did not appear to have the lipid accumulation which was so prominent AMERICAN

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in

titit.

i.t\t’.

I htm ;IIT

I‘hese

t\\‘(J \ .triants

tiifferences of Farber’s

suggest disease.

that

A, /;nr,ic’//~/(~r/lc’)lt: \Ye wish to thartk Dr. (;~~r;~ld \\‘. H,I/;II tl 01 Hyannis, hlassachusetts, this lxitient to us; Drs. Julian N. for I dei-rilig K;mtc~ ;lntl Ko4coe 0. Brady, Neurochemistry Institute of h’eurologiLabot~;lLcll-\ ( S;ltional t al I)iscz,tse\ .1nt1 Blindness, Bethesda, Ilarylatltl. 101. 1)crlorming assays of ceramiclxc, .~ntl ~])hingornyelinase activity cerebroGtl,~~e and IOI- l)rovitling tertain glycolipicl standal&; ant1 111. John Sh1110 for his assistance in thcrminilig Ihr ceralnitle com[)osition.

1

2 3

4.

5

6.

i,

8.

9

10.

11

12.

13.

“01.

S.. C.nrrrx, 1, and UZAIAN, L. L. Lipogran~~lomatouis. .4 new lipo-glyco-protein “storage” cliscarc,. /. .llt. \inni Hosp., 24: 816, 1957. /rn I R~L ~6~1, R. Disseminated lipogranu~omatosis ~,Faibn-‘s (llseahe). lctn paedint., 47: 501, 1958. \Is[.I.-11 \l. \. K.. \f.\RTZ, D. G., Dor;cus, W. F. ;111(1 (.FFPLR~. 1.. J, Farber’s disease. Report of jl c.~se with ob\eI\ations on its histogcnesis and I)OI<‘Y on the natlIre of the stored material. /. I’c,tlirrt.. ti1: ““1, 1962. SCI~.\S(:HE. ~1. F.. 1511RVAN, S. M., SOPHER, R. L. an<1 0’1 01 GHI IN, 1%. J. Disseminated lipogranchanges. ulomatosis: roentgenographic tXll\ Rntliol~~~r-\. ,S!?:(iii, 1964. . (:o(;.zu. 1;: (... KL!L.%BARA, T., MOSER, H. and [I \?\KI). (,. \V. Retinopathy in a case of Farber’s IipogI ;tnIIlomaIosis .4rch. Ophth., 75: 752, 1966. (:Ro(:hlR. \ C.. C:OIIFK, J. and FARRFR, S. The “lil~~l~~aIllllr~~~~~lt~)~i~” syndrome; Review, with Ivpor~ (lf l)atient \ho&ng milder involvement. In: IIIIKIII~ Disoltlc~Is of Sphingolipid Mctabolism. 1). ~I,%. Etlitctl bv Aronson, S. M. and Volk, 1%. \\’ Oxfonl. 1%;. Pergamon Press, Ltd. 1~ \vI’I\I. 5. .111tl C:r \I SEN, J. Farberschc Krankheit (tli~scminIcr1~ L.ipogranulomatose) Hel7wt. ~wtlirrr. c,c/a. 2: 500, 196i. PruuI .\. Cn.E. Histochemistry, Theoretical and %
,I;.

“1 <:L\IBI‘R

1969

16. Rousl,~, C., 41.\KOTOS. .\. A. ;ltld 1.1 IICC.IIIR, S. Quantitative analysis of l~hosl~lIolil~ic!~ 1,) thin layer chromatography atId phosphoIl~\ .rIlal\sis of spots. LiPids, 1: x.5, 1!)66. 17. SCHN.~IZ, J, D. ~\utomatic titr-atioll of tlce f.Jtfy acids. J, Lif~id. IZes., 5: 483, 1964. 18. S0rIl.I I R, S., CI OVER, (;. .\. Xld 1klR1 11 \\, ‘2. 2% method for the separation of aci(l nI~Icopol~sa~charides: its application to Ihe isolation of hcpatin from the skin of eats. ,I. Niol. (:/,crr,.. 2.36: !I83, 1961. 19. 1)1 FFRRANTE, N. and RICH, (1. ‘Ihc, (l~~Ic~~tnit~l;rtion of atid aminopolysaccharide in ulinc. 1. ln6. ti C/ill. 1!&?d., 48: 491. 1956 20. DISCIII, %. h new specific color IcactioIl of hcuu*onlc acids. /. Iliol. C/,er,r., 167: IX!I. 1947. 21. PRLNSKI, ,I. I,., FI-RREIRA. Cr., CARR. S. ;IIN~ Xlosr~, H. W. Ccramidc and gangliosidt a(( umltlation iII Farber’s lipogranulomatosis. 1’1oc. \oc. /k/wr. /colipitls in metachromatic lcucodystrophy. I. .Vru~ oclrer,r., 13: 103i,

1966.

30. KIs~r.\roro. hydroxy

Y. and R,uns, 5. S. Occurlcnce of 2. fatty acids in animal tissues. 1. Lipid Res., 4: 139, 1963. 31. KOREA, S. R. and GONATAS, J. Separation of human brain gangliosides. Life SC., 1: 296, 1963.

890

Farber’s

Lipogranulomatosis-Moser

32. SUZUKI, K. The pattern of mammalian brain gang liosides. III. Regional and developmental differences. J. Neurochem, 12: 969, 1965. 33. SLEIN, M. W. D-glucose. Determination with hexokinase and glucose-6-phosphate dehydrogenase. In: Methods of Enzymatic Analysis, p. 117. Edited by Bergmeyer, H.-U. New York, 1963. Academic Press, Inc. 34. SWEELEY, C. C. and WALKER, B. Determination of carbohydrates in glycolipides and gangliosides by gas chromatography. Anal. Chem., 36: 1461, 1964. 35. GATT, S. Enzymatic hydrolysis of sphingolipids. I. Hydrolysis and synthesis of ceramides by an enzyme from rat brain. J. Biol. Chem., 241: 3724, 1966. 36. AUSTIN, J. H. and LEHFELIYT, D. Studies in globoid (Krabbe) leukodystrophy. III. Significance of experimentally-produced globoid-like elements in rat white matter and spleen. J. Neuropath. & Exper. Neural., 24: 265, 1965. 37. MOSER, H. W. and LEES, M. Sulfatide lipidosis: metachromatic leukodystrophy. In: The Meta-

38. 39.

40.

41.

42.

et al.

bolic Basis of Inherited Disease, p. 539. Edited by Stanbury, J. B., Wyngaarden, J. B. and Fredrickson. D. S. New York, 1966. McGraw Hill, Inc. Ba+nu, R. 0. The sphingolipidoses. New England J. Med., 275: 312, 1966. KANFER, J. N., BLUME, R. S., YANKEE, R. A. and WOLFF, S. M. Alteration of sphingolipid metabolism in leukocytes from patients with the C$h;dia2k;gHi~ls0hi s6ydrome. New England J. : , . FELDMAN, G., FELDMAN, L. S. and ROUSER, G. Occurrence of glycolipids in the lens of the human eye. J. Am. Oil Chem. SIX., 42: 742, 1965. SVENMERHOLM,L. Quantitative estimation of sialic acids. II. A calorimetric resorcinol-hydrochloric acid method. Biochi?rl. et biophys. acta, 24: 604, 1957. BIERMAN, S. M., EDGINTON, T., NEWCOMB, V. D. and PEARSON, C. M. Farber’s disease. A disorder of mucopolysaccharide metabolism with articular, respiratory and neurologic manifestations. Arthritis e!r Rheumat., 9: 620, 1966.

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