}N'J,.: HN;\" IONAL JOU!lN;\L OF 1 .EI'ROSY Volume ~8, Number ~ Printed in tilt: U .S.A. Lipids in Leprosy 2. Histochemistry of Lipids in Human Leprosyl,2
Isamu Sakurai and Olaf K. Skinsnes 3
It is well known that lepra cell s contain stain (:1 1). 10 per cent neutralized formalin "large amounts of lipid substances, especial for various stains useful in the identification ly phospholipids and fatty acids. Opinions of lipids, and formol -calcium solution for as to the origin of these lipids have, howev Baker's and Klti ver-Ba lTcra's methods for ,er, been varied ( 1.8, H. 1(1 . 17, 2S- :l2) . phospholipids. This progressive accumulation of fatty Each of the tissues fix ed in Hic1ley's solu ~ ub s tan ces in lepromatous macrophagcs tion was transferred to 70 per cent ethyl and the absence of such accumulation in alcohol, dehydrated in graded alcohols, tuberculoid leprosy warrants continuing in embedded in paraffin and cut at four mi vestigation with respect to its relationship crons. Tissues fi xed in either 10 per cent to the apparent defect in cellular immunity neutrali zed formalin or formol -calcium so that is characteristic of lepromatous lepro lutions were embedded ,in carbowax (poly sy. The following obscrvations are a contin ethylene glyeol, Union Carbide Corp. ), uation of prcliminary investi gations initi which docs not interfere with the various ~ ted in this area. lipid stains (2~) . They wcre cut at 4 to 8 microns in a low humidity, air-conditioned MATERIALS AND METHODS room at a temperature of approximately Skin biopsics from 21 Hong Kon g and 22°C. The sections were then placed in a Taiwan Chinese patients with leprosy were flotation bath made of 40 volumes of di divided into three groups: ( 1 ) 14 cases of ethylene glycol, 50 volumes of distilled histologically lepromatous leprosy, either water and 10 volumes of 40 per cent untreated or treated with diaminodiphenyl formalin to dissolve the embedding mcdi sulfone or Giba-1906, (2) two cases of um and to spread the sections. They were histologically tuberculoid leprosy, and ( 3) then picked up on glass slides precoated fi ve instances of leprosy treated with B.663 vvith a thin £1m of gelatin eonsisting of a , for from two months to over a year. The mixture of JO gm. gelatin, 60 ml. distilled latter group included two cases of lepro water, 50 m1. glycerine and 1 ml. phenol. matous and three cases of intermediate The mixture had been preheated to dis ( dimorphous) leprosy. The B.663-treated solve the gelatin, and filtered. cases were regarded as a distinct group Histochemical procedures were per because reRections of cellular lipid metabo formed mainly in accord with the methods lism in these p atients have characteristics detailed by Pearse (]9) and Okamoto et differing from that in the other groups. al. (18). The paraffin-em bedded tissues The spccimens were immcdiately fi xcd were sta in ed with hematoxylin and eosin, in three different fi xatives: Ridley's periodic acid-Schiff, Ziehl-Neelsen's stain, modified Zenker-formol solution for Triff Triff stain (:H) , Sudan black B stain eom bined with lipid extraction by ethanol, pyr I R ccei,'cd for publication 9 April 1970, idine. or chloroform-methan01 (2: 1 ) mix ~ This work was supportcd by Grant AI·088i8 ture for firmly bound lipids and ceroid. from the 01a li oll al Inslitute of All ergy and Infec· ti ous Diseases, National Institutes of H ealih, Be· Sudan black B stain together with extrac Ihesda, ,\Iaryl:lIld ~OO I 4. tion by acidified chloroform-methanol mix :1 I. Sak urai. M.D., SeD., Assi stant R esearcher. ture ( 1% conc. hydroehloric acid in chloro and O. K. Sk illSn es. M.D ., Ph, D., Departmen t of Pa thology, Uni"crsity of Hawaii School of Medicine. form -methanol, 2: 1, mixture ) was used as a Leahi Hospital. 3675 Kilauea Avenue. Honolulu, negati ve control for the latter procedure. Ilawaii 96816. (D r. Sa/ntraj's presenl nrlrll'ess: Dc· partment of Pathology, N ihon University Sc hool of The earbowax-embedded tissues were sub ,Medicine, ltabashi.ku, Tokyo, Japan. jected to the following battelY of pro- 389 390 Internati01U11 J oumal of Lepmsy 1970
Sudan III & Nile Blue Neutral Sulfate No . of Fat Red No . of Fat ~ases cases
orange- pink-blue Lepromatous 14 10 yellow or blue -"'+ Tuberculoid 2 trace 2 blue -
mostly mostly B.663-treated 5 5 red red t--v-H+-
FIG. 1. Histochemistry of neutral lipid in human leprosy. cedures: Sudan III stain (Daddi's meth than neutral fat such as cholesterol es ters, od) for lipid in general; fett rot stain for phospholipids and fatty acids may bc lipid in general; Nile blue sulfate method stain ed orange-yellow, and glycolipids arc for neutral fat and lipoid; Fischl er's method not generally stain cd with Sudan III. Neu for fatty acids together with lipid extraction tral fat should stain red, cholesterol s are by pyridine as a negative control; Okamo pale red and oHier lipoid substances such to, Shimamoto and Sonoda's methods for as phospholipids are blue by means of Nile cholesterin and its esters; Okamoto, Ueda, blue sulfate method (18, 19) . As shown in Kusumoto and Hashimoto's m ethod Figure 1, lepra cells in lepromatous leprosy (modified Molisch reaction ) for glycolip are stained orange-yellow by Sudan III and ids; Okamoto, Shimamoto, Ueda, Kusumoto fett rot stains, and blue or pink-blue by and Shibata's mercury diphenylcarbazone Nile blue sulfate, suggesting that some lep method for phospholipids and glycolipids; ra cell s contain a small amount of neutral Baker's acid hematein method for phospho fat, but the main components are probably lipids, together with Baker's pyridine ex lipoid substances as first pointed out by traction technic as a negative control; M itsuda (16) . In contrast to lepromatous Kluver and Barrera's phthalocyanin method leprosy epithelioid cells in lesions of tuber for phospholids excluding sphingomyelin, culoid leprosy present only trace of color in and probably glycolipids. both Sudan III and fett rot stains, and are stained light blue by Nile blue sulfate. RESULTS Tuberculoid leprosy lesions contain only Neutral fat (Fig. 1). Three methods were small amounts of lipids, which can scarcely employed for the identification of neutral be demon strated by histochemical proce fat as well as lipids in general. These dures. consisted of the Sudan III, fett rot and Four of fi ve cases treated with B.663 Nile blue sulfate stains. There are no spe showed red reaction in lepra cells by the cific histochemical methods available for Nile blue sulfate method, and were stained neutral fat. The Sudan and fett rot dyes red by Sudan III and fett rot stains, in stain not only neutral fat but also other stead of orange-yellow as seen in untreated lipids. The presence of neutral fat in tis lepromatous leprosy. One of these five sues, however, may be suggested by a red cases yielded almost similar results to that color reaction with Sudan III and fett rot found in untreated lepromatous leprosy. as well as by metachromasia (pink-red) in This case had been treated for the shortest the Nile blue sulfate method. Other lipids period among five cases. Most macrophages 38, 4 Sakurai & Skinsnes: Lipids in Leprosy. 2. 391
Number Fischler's of Leprosy bacillus cases method
Lepromatous 14 -H+ many
Tubercul oid 2 -- ,-...../ + none or few
B.663-treated 5 - -.,J + few
FIG. 2. Histochemistry of fatty acid in human leprosy.
(lepra cell s) in B.663-treated cases have themselves stained with this meth od and larger vacuoles or globules in their cyto th e bacilli had bcen destroyed and digcsted plasm which proba bly contain la r ger in the tuberculoid lesions. Largc globi in amounts of neutral fat th an in untreated lepromatous lesions stain ed dark blue. lepromatous tissues. B.663-treated cases varied in rcacti on to One exception was found in the un fatty acid staining. Two of five cases were treated lepromatous group. These sections slightly positive and the other three were reveal ed nonspecific panniculitis in reac negative. Positive cases presentcd granular tional phase. The color reaction in lipid form s of bacilli which are stain able with staining was peculiar in this case, differing this method. The positive results were from that in the other cases of lepromatous reconfirmed by pyridine-extraction. leprosy. Some macrophages had a great Glycolipid. Okamoto, Ueda, Kusumoto deal of neutral fat, demonstrating as bright and Hashimoto's modified Molisch reaction red in Sudan III and fett rot stains, and as was employed for the identification of gly pink-red in the Nile blue sulfate method. colipids. All cases in the three groups . Such neutral fat-laden cells are commonly studied were negative by this method . seen in the deep corium and subcutis, or Phospholipid (Fig. 4). Phospholipids were around the dermal appendages. Besides evaluated by three different methods con lepromatous changes, this case showed lep sisting of Okamoto et oZ. mercury di ra reaction manifested as nonspecific pan phenylcarbazone method, Baker's acid he niculitis, vasculitis and less bacilli. Neutral matein method, and Khi ver and Barrera's fat deposition in macrophages in this case copper phthalocyanin method. The results may have resulted from phagocytosis of are shown in Figure 4. necrotic fat cells in the subcutaneous tissue. Baker's method seemed to be the most Free cholesterol and cholesterol ester. sensitive. Positive reaction was clearly Sections were strained with the Oka shown as dark blue against a yellowish moto et aZ. sulfuric acid-acetic acid and brown background (Fig. 5 ), whereas in the sulfuric acid methods (18, 10) . All the other methods a positive reaction was cases studied are negative to both methods: presented as darker tone in a lighter back Fatty acid (Fig. 2). Fischl er's method ground of the same color so that evaluation was employed for fatty acids. As shown in was relatively difficult. Figure 2, all of the 14 cases of lepromatous The results of phospholipid-histochemis leprosy were positive for fatty acid staining try indicate that lepromatous leprosy mac (Fig. 3), whereas most cells in tuberculoid rophages yield a strong reaction in most lesions were negative. Leprosy bacilli are instances, whereas tuberculoid lesions are Tntemntional Journal of Leprosy 1970
v . .,. ~ It , .. .. ,'" • • ,.. 11;, ... '\ • "" .J ..
• 'II•
FIG. 3. Lepromatous leprosy. Fischler's stain for fatty acid. Originalmagniflca'ti on XIOO . negative. It is interesting that B.663-treated tion has been designed in this laboratory. leprosy vari cd betwccn ncgative and weak After extracti on of paraffin- embedded ti s ly positive. sues by a chloroform-methanol ( 2: 1 ) mix Firmly bound lipids (Fig, 6), It is well ture, most lipids should be removed from known that certain kinds of lipid are not the tissues but firmly bound lipids should dissolved in ordinary fat solvents such as remain. They can be visualized by Sudan ethanol, methanol, chloroform, acetone or black B stainin g. If positive by this method, pyridine, because they are firmly bound to the firml y bound lipids should be removed proteins or polysaccharides. These can be when extractcd with acidified organic dissolved in acidified or alkalized solvents. solvents. This can be used as a negative Such lipids have been termed "firml y control. Accordingly aftcr extraction of bound lipids." On the basis of such chemi paraffin-embedded ti ssues with a chloro cal characteristics of firmly bound lipids, a form-methanol (2: 1 ) mixture, the sections histochemical method for their identifica- were stained with Sudan black Band COlln- 38, 4 Sakurai & Skinsnes: Lipids in Lep7'Osy. 2.
Ok allOto c t . 1 IIC thO
• 01.:1.oto (' t a 1 .cth OtI ; COllOlllOn ...· thO
FIG. 5. Lepromatous leprosy. Baker's acid hematein method for phospholipid. Origin al magnification XIOO.
DISCUSSION cholesterols in lepra cells. According to sev eral subsequent studies phospholipids and Lipid composition of lepra cells and its fatty acids are regarded as major lipid com origin. The lepra cells were first named by ponents of lepra cells (Fig. 10 ). There are, Virchow, in 1863, (33) who noticed that however, some differences in concepts re these cells characteristically had a tendency garding the nature and origin of stored to show vaculoes. These he thought to be lipids. Some investigators have held that the result of hydropic degeneration. Unna neutral fat is one of the important com in 1898 (32) suggested on the basis of osmic ponents of the cell lipids (10, 13, 17). Some acid stained tissues that the vacuoles con report that cholesterol is contained in the tainedlipid substances, and Mitsuda in 1918 stored lipids in advanced stages of lepro ( 1G) reported the presence of a certain kind mas (2D,3 0, 31), while others deny the pres of lipoid (phospholipid or glycolipid ) and ence of lepra cell choles terol (9, 11,12), There fatty acids, and absence of neutral fat and seem to be three different views regarding 38, 4 Sakurai & Skinsnes: Lipids in Leprosy. 2. ,'395
Number Sudan hlack R stain Nega tive Control Firmly of after e xtracti on bv aft er extraction by bound cases ch l oroform-methanoi ac idifi ed chloroform- lipid methanol
Lepromatous 14 - '" + -- --'+ Tllh er cllloid 2 - ,-.,.,,/ + -- -"'-/+
R. 6 (\ ~ -treated 5 "-" +tt- - ~ +(acid-fast ness) ± -+++-
FIG. 6. Histochemistry of firml y bound lipid in human leprosy. the ongm of the stored lipids (28) . One rous infiltration (10. 17). Most observers, holds that the lipids a r e d e r i v e d however, seem to hold that both factors, from bacilli phagocytosed by the macro- bacilli and lipophanerosis, may playa rolc phages (11 , 12, 16) , Another maintains that in the origin of lipid storage in lepra cells these lipids originate from fatty degener- ( 13, 29, 30, 31), Davison et aZ. ( 8 ) and ation (lipophanerosis) occurring in the cy- Ghosh et aZ. ( 11, 12), pointed out that toplasm of lepra cells (1). A third view is tuberculoid macrophages present only min that the lipid storage results from phagocy- im al lipid staining whereas lepromatous tosis of fat droplets by macrophages. These macrophagcs have a great deal of aceumu fat droplets are believed to result from lated lipid substance. It is evident that destruction of th e subcutaneoll s fat by lep- lepromatous macrophages readily phagocy-
-...,): ... • ,.:t".. Jr' /..., .. .. : ) v-.. . . '"
FIG. 7. B,663·treated leprosy. Sudan black I3 stain (left ) and Ziehl-Neelsen stain (right) after lipid extracti on, TIl ere is a sudanophilic acid-fast substance around the rim of vacuoles in lepra cells, which is insoluble in fat solvents. Original magnificati on XlOO. 396 International Journal of Leprosy 1970
Lepromatous Tub e rcu l o id R. 663- trea ted
A'I I l. ipids pos it ive trace positive
Neutra ) f a t -'V+ -- -tt+ ra t ty acid -ttt -""'+ - "" + Cho l est ero l ------~ its ester
r. l ycoli pid ------
Phospho l ipid itt -- - --+ Pi rm l y bound lipid -""+ -....,+ -ttt I. e pros y hac i II us -H+ -,....,,+ + F IC. 8. Lipid histochemi stry in human leprosy.
FIC. 9. Paraffin -embedded tiss ues of two cases of B.663-treated leprosy (upper row), compared with DDS-treated (left lower) and untreated (ri ght lower) lepromatous leprosy. B.663-treated leprosy presents a pigmentation throughout the dermis even after lipids and lipophilic B.663 have been removed from tissues by fat solvents. 38, 4 Sakurai & Skinsnes: Lipids in Leprosy. 2. 397
\'ErrrRAI. FA1TY (JIO LESTERO L GLYCO· PIlOSPIIO · LIPO- FIRMLY SOUSD LlPtD ORtGtN r ,\T ACIIl FREE I ESTER I.lPIO LIPID PROTF. I~ LlP t D l ~lfT<;!rnA . 1911'1 - BACt LLI haci+ 11 i lipoid
BACt lLl ~ 1rr.[)A, 1 ~ · 1 8 + CHl DEGENERATION ± - - ..... t l ecithin
BACtLLI & H ·\I ~·\ O ·\ • 1955 - -+ + CELL OEGE~ER ATJON e3rl v Jnt (' + + caTt y
BACILLI \ 95R & <'; llk ", + - r.J + CEll. DFGENERATION - --+ h;lei I ! i + l cci t hin
·\I~'U:, \I" ·\. 19SR CELL DEGENERAT tON
L1 POPROTE I N ,'tArn,\, 1960 + FRO!>I ALooD s aturated + + + FIII\II,!!I, 1!16n + UPOPIIAr.lA
GI1 O'> I! , 1~ l(12 + BACt LLt ± had I I i +
S HIIR\! I. Melt!.l & 0..; 1:1\<.;\ 1", rt=u. OFr.E~FRATlO~ h ; I+~' I I - "'t -"'+ i i + F IG. 10. Histochemistry of lipids in lepra cells and their origin .
tose M. leprae and many of the phagocy and that neutral fa t and cholesterol, both of tosed bacilli have undigested, lipid-ri ch cell which begin to appear in advanccd stages, walls. Figure 10 compares the present may possibly result from lipophanerosis of study with those of others. The findin gs are macrophages. similar to those previously reported by Compari son with lipid staining in tuber Veda (3 1) , Harada ( 1:{) , Sugai (~D) and culos is and suppurative lesions is contribll . Ghosh et al. ( 11 , 1 ~) , except for choles bve to understanding. Kusumoto (H\) terol, whi ch was shown by Veda and Su gai stated that early tuberculous granulomas, but not identified in this study. Fite (9) without caseation, contain only minimal fa t ' indicated that cholesterol was not present substances, mainly phospholipids, At this in earl y stagcs of lepromas, and Harada stage fairly large numbcrs of M. tuberculosis (.13) stressed that neutral fat tends to be are usually present. As lesions progress and present in lepra cells with large fat globules caseation necrosis develops, lipid staining or globi in advanced stages of lepromatous becomes more intense and the bacilli in the lcprosy, Also from electron-microscopic lesions decrease in number. In advanced studies it has bcen suggested by Ya mamoto tuberculous granulomas having caseation et a1. (36) and Imaeda ( t4) that thc necrosis, neutral fat and cholesterol, and foamy structurc of human lepra cell s is a phospholipids are f 0 u n d m a i n I y in terminal phase of intracellular structural peripheral areas of caseation nccrosis ad changes. H istochemical methods for fatty jacent to the surrounding granulomatous acids and phospholipids stain the mycobac zone, Lipids in nonspecific suppurative tt' ri al cell wall s on bacteri al smears (5 , 24) . lesions arc mainly composcd of neutral fa t Dharmendra also demonstrated that phos' and cholesterol fat whereas those in tuber phoIipids were contai ncd in M, leprae (7) . culos is are mainly phospholipids ( Fig. 11 ), Thus fatty acids and phospholipids are Thus it seems reasonable to think that major lipid components of M. leprae. It phospholipids are probably the main ex seems reasonable to think that the stored ogenous lipid component of reti culoen lipids, especially fatty acids and phos dotheli al cell s in lesions caused by myco pholipids, are mainly of bacillary origin , bacterial infections. The findin gs reported 398 lntem atiorud Journal of Leprosy 1970
NEUTRAL FATTY CHOLESTEROL GLYCO- PHOS PHO- TUB ERCULOS IS FAT ACID LIPID FREE ESTER LIPID BACILLUS
M EARLY TUBERCLE T I - - -- U L WITHOUT CASEATION ± + -tH- B I E A R R TUBERCLE WITH C Y - - - -tt U CASEATION + L 0 P * * S R EARLY TUBERCL E - I 0 - - ± + S D U * * C TUBERCLE WITH T - I ~1ARKED CASEATION -it - + itt -ttt- ± V E
SUPPIiRATIVE L ES IO~: - - ABSC ESS G GA~GRENF. * + - + FIG. 11. Lipid histochemistry of ,tubercul os is and suppuration. by Kusumoto for other inRammatory condi outlin ed above. Further studies on lipids in tions also seem to support the concept that leprosy by chromatographic analyses are in neutral fat and cholesterol in macrophages preparation (26) . may result from cell degeneration, and B.663 pi gment~tion. Leprous lesions hav phospholipids may be derived from myco ing the peculiar pigmentation caused by bacterial cell walls. B.663 treatment show yellowish-brown pig Chromatographic analyses by a thin ment in the cytoplasm of macrophages, layer technic done recently in this laborato which can be identified in thick unstained ry (26) have shown that all major lipid or methylme blue stained paraffin-sections fractions such as phospho- and glycolipid of tissue at 10 to 20 microns, even after fractions, free cholesterol, free fatty acid, B.663 and most lipids have been removed triglyceride, methyl ester of fatty acid and by organic solvents (2:3). Since B.663 is a cholesterol ester, are identifiable in purified red dye, abnormal coloration of the skin M. lepraemurium suspensions as well as in can be produced by a deposition of the both human and murine leprous tissues, as drug itself. In addition, however, there shown in Figure 14. Murine leprosy bacilli must be other pigment than the drug in the contain large amounts of lipoid substances leprous macrophages which are not dis (phospho- and glycolipid ) and free fatty solvcd in crganic solvents. acid, whereas murine leprosy tissues have We have noted (25) that a rabbit fed more free cholestcrol than do M. lep 13.663 (50 mg.Jkg. for 5 days) in vegetable praemurium and normal skin. This indi oil, at autopsy showed that its fat tissues cates also that accumulation of free choles had an usual yellow tint which turns to terol in the tissues is probably caused by orange-red shOltly after exposure to the air. tissue lipophanerosis. Murine lepromas and These fat tissues, however, retain their nor human lepromatous tissues contain more mal appearance if they are kept in a nitro free fatty acids and lipoid substances gen gas atmosphere with exposure to sun (phospho- and glycolipid ) than control tis light but without exposure to the air. B.663 sues or tuberculoid leprosy lesions. Both is dissolved in oil or organic fat solvents. free fatty acids and lipoid substances are but is not water-soluble. This sugge~ts that also richly contained in the bacilli. These B.663, a red dye, becomes colorless after results also support the concept concerning absorption through the digestive canal, is the origin of stored lipids in lepra cell s deposited in fat cells and may again be- 33, 4 Sakurai & Skinsnes: Lipids in Leprosy. 2. 399
PIGMENT IN CEROID B.663 TREATED
NATURAL COLOR yellow brown yellow brown rim of fa! olobules rim of fa! olobules LOCATION of phaoocy!es of lepra cells
WATER insoluble insoluble
FAT SOLVENTS i nsol uble in solu ble BLEACHING + _ 2hrs + 2 4hrs IRON REACTION + + GMELIN R.
STEIGER R. ACID FASTNE SS + + HUECK METHOD + + SCHMORL R . + + CHROME AWM HEM . + + PAS + + FIG. 12. Characteristi cs of pigment in B. 663-treated Jeprosy. come red on oxidation. Intraperitoneal in- in alcohols and chloroform which extract jection of B.663 dissolved in vegetable oil the drug from the ti ssues (23). (25 mg. in 0.5 m1. of oil ) into mice infected In various histochemical stainings, B.663- with M. lepraemurium resulted in a red- treated leprosy is significantly different di sh-brown or reddish-black pigmentation from untreated lepromatous leprosy. The limited to nodular granulomas in the peri- B.663 treated lesions contain more neutral toneum by eight 'weeks after injection fat and firmly bound lipids, and lesser (25) . Peritoneal spread preparations and amounts of fatty acids and phospholipids frozen sections of various organs such as ( Fig. 8). Leprosy bacilli are far fewer in li ver, kidney, lung and spleen showed dark the lepra cell s in the B.663 treated and are . red, elongated cylindric clystals. These granular. Histochemical characteri stics of crystals were completely di ssolved and re- the pigment in macrophages of the B.663- moved from the ti ssues by alcohols, and the treated lesions are quite similar to those of pigmentation disappeared after extraction ceroid pigment, as shown in Figure 12 (23). with fat solvents. On the basis of these Most macrophages in the lesions of B.663- results and other reports (2, 3, 6. 27, 35), it treated leprosy have large fat vacuoles in seems evident that the early stage of red the cytoplasm, which are fill ed with neutral pigmentation is due to a deposition of fat and the rim of which is made up of acid B.663 itself in tissues, mainly in fat cells, fast, sudanophilic ceroid-like substance that and that the deposited drug can be re- is insoluble in ordinary fat solvents (Figs. moved by fat solvents. However, in human 7 & 13 ). Some investigators have proposed lesions of patients treated with B.663 for a melanin-theory for late B.663 pigmenta- several months or for over a year, there h tion (4) . Late B.663 pigmentation, owever, appear to be other pigments than the B.663 is usually seen also deep in the corium, present in macrophages which are not re- moved by organic solvents. This is ye I- where melanin is not generally presentI ex- lowish-brown and usually located around cept in certain abnormal conditions suc 1 as the rim of the fat globules in macrop Il ages. blue nevi or Ota's nevus. As shown in Figure 9, pigmentation still Since ceroid is a lipid derivative (20) , it remains after tissues have been processed may be that this pigment is produced from 400 International Journal of L eprosy 1970
LEPRO~lA.TOU S LEPROSY B.663-TREATI:D
LEPROSY BAC I LLlIS Nm1EROUS, SOL ID A FEW, GRANULAR / / ,
NEUTRAL FAT, ./ } ) MAIN LTPTOS PHOSP HOLIPID, FATTY ACID FIRMLY BOUND LIPID YELLOW BROWN, PIr.~ t ENT ACID FAST, SUDANOPHILIC I NS OLUBLE IN FAT SOLVENTS
FIC. 13. Diagram of histochemical characteristi cs of lepra· cells in B.663-treated leprosy as compared with untreated leprosy.
• M&1'JHL ESl'KR or ".~ ': ·r~ F~·7'/:'l Cf Fl'!Y M: m • '>oAT .. A'~ll}
' FA,.,.Y Aero ,
, "11'6;£ CHOLE TS:ROl I,REI! CHOI.IIS~
G~YCOL 1Plll .. ~ PhOSPHO: lPm CLYCOL IPm ... !lV?TNE " liRIN": P!\CIl P"~lpm S~l~ SKIN CO\-rROl LEPROS Y liOl1!!AL LEPR(Jo!ATOtS TUBERClILOlD 1 SKIN LEPNa; Y LEPpa; Y ;;j ,
FIC. 14. Separation into major lipid classes by thin-layer chromotography in lvl. leprael1wrium, and murine and human leprosy tissues. Thin-layer plate : Silica gel H. 250 mi crons thick (Analtech ) . Solvent: Petroleum ether-diethyl ether-acetic acid (90: 10: 1). Detection: 100% sulfuric acid with charring. 38, 4 Sakurai & Skinsnes: L ipids in Leprosy. 2. 401 acid-fast, lipid components of bacilli which RESUMEN have been altered by acti on of the drug. Utilizando metodos h is t o quim icos, se The released acid-fast components of my efectuaron estudios de los Iipidos de material cobacteria may be bound with substances de biopsia de pacientes con lepra lepromatosa which are insoluble in fat solvents, as sug ( 14 casos), tubercul oide (2 casos) y lepra gested by Reeves and Anderson (21). lepromatosa tratada con B.663 (5 casos). La iepra lepromatosa presenta tincion intensa de Problems in the field of lipid histochemis lipidos mientras que la lepra tuberculoide try. As shown in Figure 14, our chromato revela sola'mente una tincion minima. Las graphic studies indicate that all major lipid celul as de lepra en la lepra lepromatosa con fr actions arc present in the bacilli as wcll as tienen muchos fosfolipidos y acidos grasos y in leprous tissues, although cholesterols are solo pequefi as cantidades de grasa neutra. En not identified by histochemical methods. este estudio no se encontraron coles teroles. Histochemical methods for certain lipids Los fos folipidos y acidos grasos alm acenados are not sensitive enough to identify small probablemente se ori ginan de los M. ieprae amounts of lipids. However, histochemi stry fagocitados. La grasa neutra puede se r con has some advantages over bi ochemi cal an secuencia de lipofanerosis. Los casos de lepra alysis. In identifying chemical substances tratados con B.663 presentan un pi gmento by color reacti ons within cells or tissues amarillo-marron en los macrOfagos de las lesiones de lepra, que es similar en sus there is no sacrifice of morphologic observa caracte risti cas histoquimicas al pi gmento tions, and the locations of such chemical ceroid e, ya que es in soluble en solve ntes de substances within cells or ti ssues is indi lipides, es acid o resistente y sudanofili co. En cated. Biochemical analyses of lesions often comparacion con los resultados obtenidos de include normal cellular or tissue com los analisis cromatografi cos de lipidos, las ponents in addition to the lesion material tecnicas hi stoquimicas no son 10 sufi cientemente under analysis. Combination of both meth sensibl es como para identifica r pequefi as ods of study, as here attempted, seem more cantidades de Iipidos. valuable and helpful to an understanding of the probl ems posed in the area of the lipid metabolism of lepra cells. RESUME Des etudes histochimiques des lipides ont ete SUMMARY menees sur du materi el provenant de biopsies humain es, qui comprenaient 14 cas leproma Histochemical studies on lipids were per teux, 2 cas tuberculo'ides, et 5 cas de lepre . formed on human biopsy materials which lepromateuse traites par Ie B.663. Dans la included lepromatous (14 cases), tubercu lepre lepromateuse, on a note une forte colora loid (2 cases), and B.663-treated (5 cases) lion des lipides, alors que dans la lepre tubercu lepromatous leprosy. Lepromatous leprosy lo'ide, la coloration etait tres mini me. D ans la presents strong lipid staining whereas tu lepre lepromateuse, les cellules lepreuses con berculoid reveals only minimal staining. tiennent beaucoup de phospholipides et d'acides Lepra cells in lepromatous leprosy contain gras, mais seulement de petites quantites de much phospholipids and fatty acids, and graisse neutre. On n'a pas releve de cholesterol only small amounts of neutral fat. Choles au cours de cette etude. Les phospholipides et terols were not found in this study. Stored les acides entreposes ont probablement leur phospholipids and fatty acids probably o ri gine dans les M . /eprae phagocytes. La graisse originate from phagocytosed M. Zeprae. neutre peut provenir de la lipophanerose. D ans les cas de lepre traites par Ie 8 .663, on a Neutral fat may result from lipophanerosis. observe un pi gment brun-jaun atre dans les B.663-treated leprosy presents yell owish macrophages des lesions lepreuses, sembi able brown pigment in macrophages of leprous au pi gment cero'ide au point de vue des carac lesions, which is similar to ceroid pigment teri stiques histochimiques, in soluble dans les in histochemical characteristi cs, bein g in sol solvants de graisse, acido-resistant et soudano uble in fat solvents, acid-fast and bein g su phi Ie. Quand on compare ces resultats a ceux danophilic. Compared with results from des analyses chromatographiques des lipides chromatographic analyses of lipid in leprosy, dans la. lepre, on conclut que les techniques histochemical technics are not sensitive hi stochimiques ne so nt pas assez se nsibles pour enougb to identify small amounts of lipids. identifier de petites quantites de lipides. 402 I nte mational JoU1'1lal of L e}J1'Osy 1970
Acknowledgments. A supply of crys tallin e J ,3. H AHADA, K. Histochemical studies of lep B.663 was supplied by Dr. W. Vischer, J. R rosy, especiall y th e mod e of fo rm ati on of Geigy, S.A., Basel, Switzerland. Dr. Grace lepra cell s. La Lepro 24 (1955) 277-282. Warren kindly provided biopsy material from (In Japanese, English summary) B.663-treated patients at Hay Ling Chau Lep 14. IM AE DA, T. Electron microscopi c analysis rosarium, Hong Kong. of the co mponents of lepra cells. Intel1l at. J. Leprosy 28 (1960 ) 22-37. REFERENCES 15. KUS UMOTO, Y. On the fats of the tubercll lous foci of lung. Trans. Soc. Path. Japan . 1. ARA KAWA, 1. Cytological studies on lep 37 ( 1948) 7-71. (In Japanese) rous tiss ue. La Lepro 27 ( 1958) 114- 125. I G. ~ITS UDA , K. The signifi cance of th e va c (Tn Japan ese, Engli sh summary) uoles in the Virchow lepra cells and the 2. .ATKINSON, A. J. Sl-mAGREN, J. N., BARBA distribution of lepra cell s 111 cel'tain or R UB IO, J. and KNIGHT, V. Evaluation of gans. Tokyo Iji Shinshi 2066 (1918) 1-7 , B.663 in human leprosy. Internal. J. Lep in Japanese. Tra1l slated to English : Inter rosy 35 ( 1967) 119-127. nat. J. Leprosy 4 (1936) 49 1-508. 3. BARRY, V. C. and CONALTY, M. L. The 17. OCATA, K. Some understanding of leprosy antimycobacterial activity of B.663. Lep pathology, especially the problems in rosy Rev. 36 (1965) 3-7. classifi cati on. La Lepro 27 (1958) 305- 4. BROWNE, S. G. Red and black pigmenta 312. (In Japanese) ti on developing during treatment of lep ] 8. OKAJl IOTO, K ., UEDA , M ., \ fA EDA , T. and rosy with B.663. Leprosy Rev. 36 (1965) Mizutani, A. Chapter 4, Lipids, in Micro 17-20. scopic H istochemistry, Tokyo, Igaku-shoin, 5. CAJI,rpo-AAsEN, 1. and CONV IT, J. Identi 3rd. ed., 1965, pp. 3.31-379. ( In Japa fi cati on of th e non cultivable path ogeni c nese) mycobacteri a, M. leprae and M . leprae 19. PEARSE, A. C. E. Lipids, lipoprotein s and 'I1lurium . Intern at. ]. Leprosy 36 (1968) proteolipids, Chap. 12, pp. 398-446, and 166-170. Appendices 12, pp. 687-704. In: Histo 6. CONALTY, M. L. and JACKSON, R. D. Up chemistry Theoretical alld A pplied . Vol. -take by reticuloendothelial cells of the 1. Boston, Little, Brown & Co. , 3rd. ed. rimino-phenazine, B.663. British J. Exper. 1968. Path. 43 (1962) 650-654. 20. P EARSE, A. G. E. Pigments. In : Histo 7. DHARMENDRA. The immunologic skin tes t chemistry T heoretical alld Applied. Chap. in leprosy. Part 1. The isolation of a pro 23. Boston, Little, Brown & Co., 2nd ed., tein antigen of Mycobacterium leprae. In 1960, pp. 632-680. dian J. Med. Res. 30 ( 1942) 1-7. 21. REEVES, R. E. and ANDERSON, R. J. The 8. D AVISON, A. R , KOOIJ, A. R and WAIN chemistry of the lipids of tubercle ba WRIGHT, J. Class ifi cati on of leprosy. II. The cill i. XLVII. The compos ition of the avian value of fat staining in class ification. In tubercle bacillus wax. J. Ameri can Chern . tern at. J. Leprosy 28 ( 1960) 126-132. Soc. 59 ( 1937 ) 858-861. 9. FITE, G. L. The pathology and patho 22. ROTHSTEIN, R The use of carbowax sec genesis of leprosy. Ann . New Yo rk Acad. ti ons in the histochemical identifi cation of Sci. 54 (1951 ) 28-33. lipids and other intracell ular chemicals in 10. FUKUSHI, K. and NARITA, M. Histopatho amphibian and mammalian tiss ues. Tur logical study on mechanisms of formation tox News 44 ( 1966 ) 34-37. of lepra cells. La Lepro 29 ( 1960) 15,3- 23. SAKURAI, 1. Histochemical study on hyper 154. (In Japanese) pigmented skin (ceroid-like pigmentation) 11. GHOSH, S., SEN GUPTA, P. C. and MUXER in human leprosy treated with B.663. JEE, N. Histochemical study of leproma Fed. Proc. 28 (1969) 777. tous leprosy. Bull. Calcutta Sch. Trop. 24. SAKURAI, I. and SKI NSNES, O. K. Lipids Med. 10 ( 1962) 102-205. in leprosy: I. Histochemistry of lipid in 12. GHOSH, S. , SEN GUPTA, P. C. and MUK ER murine leprosy. Internat. J. Leprosy 38 JEE, N. Histochemical observations on (1970) 379-388. tuberculoid leprosy. Bull . Calcutta Sch. 25. SAKURAI, I. and SKINSNES, O. K. Unpub Trop. Med. 11 (1963) 27-29. lished data. 38, 4 Sakumi & Skinsnes: Lipids in Leprosy. 2. 403
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