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Joyce K Newmae, PhD; A. B. Vateer, PhD; and O. 1G ReLr, PhD. Deaaer 41 Reiss•° Thu was, ineac j"9s was tt- tracted wit! `yate shakir h,re. In sor ;out after oc+ into an a« pared with I washed wit iowed by 7 qWeS of Ct Another %,jarded out -, omt of a t cordin` to THE black pigment of huitttan lung was observed and remarked upon by the end of the 18th century. In 1943, Rosen Included an excellent and detailed account of early observations of pulmonary pigmentation by many 18th and 19th century pathologials in a velume on The History. of. Mlnve Dis- eases.l Many theories were expounded re- garding the origin of the pigment; In 1847, VirdwwR auaunsuized the lwwledge to that time in a detailed papec which referred to a number of earlier theories and apressed the opinion that "das Iungenschwaez" was 'Sold blood-pigment." He analyzed some diespated material and found resemblances to b,eaaatin and gall pigments. Virchoavt and 8chetds both observed the presence of inon. In 1862, Rebsamea" differentiated the pigment in hn man lymph nodes Into progreastvely more highly oolored types, from yellow throagh brown to bladc. Later, in I866, Koedtldooffs concluded that the pigment was aoogiaous on the basis of Its placement near alveolar oell membranes. McLean,S in 1957, dealt extensively with the problem and attempted to relate- origin to intraoellular distributton. He found that all adult lungs had pigment yhen enmined under a microscope, whether or not they ex- hibited focal emphysema. The pigment was present In discreet densely black partides about 15µ in diameter. McLean concluded that some portions of the pigment found in Submitted for pubtio.tion Apcii 11,1967: .ooepted _~ ~ webt,-w.r~ ia.etwte. .wa tae dvare. ~ 1Vleand dtc oa. D env~ea ~~~ of Colorado Rep~int requests to 4200 8 Ninth Ave, Denver 80220 (Dd Newman) DEFENDANT'S EXHIBIT -T- 5~5 -•--~ ........_:~ ~nay be pr ected ~ ~ lart (Title 17 Chemical and Electron Microscopic Studies of the Black Pigment of the Human Lung the lungs of miners was bertainly exogenous; E.,Gst a Pot however, in other lungs be found black par. 1'how,d of a tides adjaoent to areas staining strongly for 1 *at 12A00 ; iron and thought these werb related to be. ~ne fins mosiderin and hence endogenous. Pratt et ;jam uad• al* explored the relation between osntrilo- ° alioes of lu bular empltiysema and the development of ca mm an- pigment deposits and found the maJorlty of I'Affuted lesions were pigmented. Reoenft while exc t )•preparatio ....:wiws ws1.1SAwal.~wa 1.a:wl..~. 4{.a e~weearms ~ ~~ i~. i-/ . __1 _I A AG- _2 t__a__ _..s a= w ww ...~..... s..b........ o..~. ..~..~...o o... .. .,.. ,..~....." ~ s..._u. .. history of ilnolang, and ooCtulational e4w `~~•' a• suc+as„ Mitct~ell and ~ showed that " ed troa. F =' tied oat.f there was a strong oort~elati~.o tt.ott between the Sam tdos presence of targe amounts of black pigment :• :thm SUM and advanced empltysema. • ~ atibited ai The general opinion expressed in the litr o[ @,e.an: erature and in text books is that the pigment lnonbatioi is exogenous elemental carbon. (The teim , Next. 1 a "elemental carbon" i8 used in thLs paper to. ~ ttism pr a..M{.. af.e .....7..... .....~.a-a t.. sar ..wnnrr+."r~.~mi1u11ier • --- :------- - -- --- ;bo1 ^ere t bined form.) Tbis study was wndertaken to ,fbe mistu r"aceunine the Mack pigment by the tech- Tbe redd niques of modera ohemistty and electron ml- : lio, and - croaoopy in the hope that more apedSc in-~ ~t formation would be obtained concerning i~ •' for 4s a,i composition and structure as well as its rda;: and ttonship to air pollutanta ~ 3nat, 'tromethal Ma 1s and Methods 1mg ~ Method of Isolatton of PiSsnentt-HntnaA,_ fi'1'he tew lunas„ obtained from autopsy, were selected ta = M~AixtllLd • use {n thts study on the basis of hish oontent of : bladc pigment Portions were sampled for light, and electron microscopy and the lungs wt". then stored frozen at -20 C until needed. Vie r initial experiments were carried out by oaitlif:vil vgatton of the lung, homo5enate in a bufferd: sucrose .olution, according to the metlwd . ~- 80111611 '.mentionc 1301aftea lrasct the i deactibe8 as foAoM c,6 Arcla Enuiron Health-Vol 15, Oct 1967 vo, 4.20- a,9 51559 0953 ~. _ ~

PIGMENT IN HUMAN LUNG-,NEWMAN ET AL 421 ~ exogenottt; .d bladc par- strong(y for dated to be. )us. Pratt et -en ceatrijo- -elopment of tne&ti* tyy ot Jy. whik pl- a preparahon was approximately 1 gm wet the preaerw ~`weight. The bits of tissue were suspended In 80 ~~~ '~ ad of 0.067M sodium phosphate, 0.45% sodium i~ eWcride, pH 7.4. An enzymatic digestion adapt- sl'w ""` betweea t~e lack pl~mettt I : . l ed in tfie 1it- : the pigment (The term this paper to z its uncom-; stdectaloett to'o by the bech-' i electt~at ati-:. e spedfic in- vn ; ~cet„itK it:%. ,e.ll,,as its reta ; .,,.; d ,o s .}; aent.-,Hamsia jtciss? The fraction with the blackest color was, in each case, the mitochondrial fraction. is was then distributed in test tubes and ex- tracted with a number of solvents with alter- pate shaking and standing at room tempera- ture. In some cases, the extraction was carried out after conversion of the centrifuged material into an acetone powder. The powder was pre- pared with 30 volumes of cold acetone, and ' .vashed with three volumes of cold acetone fol- lowed by additional washing with three vol- umes of cold ether. Another series of solvent extractions were prried out on a fraction prepared by the treat- ment of a homogenate with sonic oscillation ao- cording to the method of Hendleyio except 'thhat a Potter-Elvehjem homogenizer was used Io:tead of a blender and a single centrifugation at 12,000 X g was carried out. The final method of preparation was as fol- 1owt: under a dissecting microscope, frozen V dioes of lung were divided into pieces about 1 tcn mm and separated into pigmented and non- pitmented bits. The total quantity for each 'ed from Fitzpatrick and Hospelhornli was car- ; rbd out as follows: 1 ml of 1% aqueous oollage- `asse (ctoatridiopeptidase A) was added to the ~ titsne suspension and the preparation was in- ~ onbated at 87 C for 24 hours. Another tailliliter ft: . of the same enzyme solution was added and the Incubation was continued for another 24 hours. Next„ I tnl of aqueous pronase (streptotqyces i.grtseus proteaae, grade B) 15 to 25 units per ~ mtlh'liter was added to the mixture (4 ml etba- ;ad were added to inhibit bacterial growth) and Qte mixture was incubated 24 hours at 87 C. The residue was then recovered by ceittrtfaga-. Cou and resuspended In 3 ml of 0.1N sodium Itydroztde and plaoQd on a boiling water bath k3or 45 minutes. The residue was again oentri- fuged and washed twice with water: The insol- ttble material was suspended in 10 ml 03EM tromethamine (tRIS) hydrochloride oontain- 6L 1 mg/ml elastase (pancreatopeptidsse •E ; aYstalline) and incuba.ted at 87 C for 24 houra. 77be resulNng residue was washed twice with dhtilled water and dried in vacuo. ere adected tot.. 4 i.igh content of -. mpled for Iigbt ` be lungs were. :it needed.'T1te: out by oenerU-.: - in a buffer0.; the ethod ~ l ~ ~~ -x. ' 8olubiltty Studies.~--In addition to the above atentloned solvent extractions carried out on .bomogenates or sonicates, we attempted to ex- ~tract the dark-colored pigment from the dried ` % teaidue prepared by enzymatic hydrolysis as ~described above- These experiments were done ' as followa- bits of dried black residue and of control tissue were finely divided with scissors and placed in test tubes. The aphroprialc sol- vents were added and the tubes were heated to 60 C for several hours and allowed to cool. The heating and cooling was repeated twice. Among the solvents tested were 10% acetic acid in chloroform, 5% potassium hydroxide in rnethanol, 10% hydrochloric acid in methanol, glacial acetic acid. sodium methoxide in metha- atol, 1% sodium desoxycholate, toluene, 1; sodium dodecyl sulfate, 5% sodium hydroxide, dintetbyl sulfoxide, and 50% pyridine in water. Infrared Spectroseopy.--The infrared ab- sorption spectrophotometry was done using 1 X 5 mm potassium bromide pellets or in inin- eral o1 (NUJol) mulk. Elemental Analyses. The dry residues were analyzed by combustion, and the metals deter- mined by means of emission spectmsooPY- 7C ray Suore.oenee spectcometry was carried out on a vacuum spectrometer with a lithium fiuoride crysbd scintillation detector. Electron Mtcrosoopyr-Smau fragments from nonpigntented and plgmented portions of lung were prepared using one of the following meth- ods: (1) fixation in 4% glutaraldehyde buffered with either 0A28M sodium veronal and sodium aoetateu at pH 7.5 or 0ZM s-collidine (2,4,6 trime"pyridine) pH 7.4 at 4 C for 1 hour,la aad then postfixed 0.5 hour in 1% osmium te- troxide In the same buffer used for initial fixa- tion. 'tite tissne was rapidly dehydrated by passage Waugh a graded ethanol series and embeaded In rmdn (Vestopal)11 or epoxy resin (Epon 81lE) u Tlsstte sections mounted on coated or unooated grids were viewed with and without daldng. The sections were double stained with a 2% aqueous solution of uranyl acetate for 20 minutes followed by lead citrate for five minutes.1s Add phosphatase was deter- mLted according to the method of Daem.ti The sections wece examined with an electron micro- acope. I,IgBt Mlerosoopyr-Sectidns were prepared and stained for light microscopy according to methods desexlbed in the Manual ol the Histo- loYic and Sptdol Staining TeclutiQtees.ts The stains used for lipid were Sudan Black B, Nile Blue Sulfate and Oil Red O. 'I~e procedutes used for the detection of iron were those of Go- mod and Mallory. Tests for melanin pigment were also performed. Results and Comment EjctraeNonf--The black pigment was not soluble in any of the solvents used to extract It ftota either the homogenate or sonicated preparation. . Arch Envi.ren Health-Vol 15, Oct 1967

422 PIGMENT IN HUMAN LUNG-NEWMAN ET AL `V 3 ! Enzymat:ic Digestionr-Because of the in- solubility of the pigment, we attempted to concentrate it by dissecting out small bits of pigment-containing tissue and subjecting them to enzymatic hydrolysis in order to re- tnove other tissue constituents. Infrared spectrophotometry was used to follow the removal of protein from the pigment. After the digestion was completed, little or no amide abeorptionto was presalt. Table 1 shows the quantity of material found resist- ant to enzymatic hydrolysis In pigmented and nonpig111Bnted tissue dissected from two lungs. It is apparent that the nonpigmented tissue is almost entirely digested while 5% to 10% of the original weight of the pig- mented tissue resists the enzymatic attack. E:tractfon of Isolated Residuer--The dried residues from the enzymatic digestions were subjected to solvent extractions as de- scribed above since we thought that diges- tion of the surrounding collagen and protein might render a portion of the pigment-more accessible to solvent action but again no no- tioeable portion of the colored material dis- solved. Combustion of the Residue. Three different lungs were selected for enzymatic digestion and subsequent combustion anal,y- sis. The selection was on the basis of dif- ferenoes in appearance by electron mi(x+o- scopic observations (see below). (Only three were processed because the dissections were very long and tediaus.) Table 2 shows the proportions. of the organic or combustible fraction to the Inorganic or nonoombustible residue.- The composition of the pigmentB from the three lungs differ greatly. As shown in Table 3, some similarities can be noted in the combustible fractions of lung samples C- 30 and C-47. The sample prepared from the third. lung (C-44), however, contained a higher percent of carbon in its combustible Z Tabte 1.--Recovery of Pigment After Exhaustive Enzymatic Digestion' •For digestion procedure see text. tMeasured in mg dry weieht/am wet weight. Li Table 2.-Composition of Human Lung Pigment I ;with t h er ..~~....~..~_ u...,.,....h....sw. , Ot Fraction• Residuet, icall;/ LunQ No. 'X % were t C-3o 79.3 20.7 tative C.44 64.9 35.1 ' Dolloe' C-47 39.5 .. 60.5 thesis: •Csiculated as follows: mg starting materlal -m` non- eombustiae restdue/mi startini material X 100. llRs ow: me nddue/n~ b starting at 900 C calculated as to ng material X 100. (0.151 0.85i~ (0.02: :1er ( fraction. If one assumes only part of the car- to 0.( bolt in this sample Is organic and adjusts the dirc=- value for cbemically combined carbon to and b3.8°Jo, then one obtains a value for hydro- 44 (G gen, -nittogen, and the balance of 9.0%, found 4.4% and 83 0°,J'o respectively. These values anioul are in reasonable agreement with those found In the other two preparations, and suggest a fairly constant composition for the organic portion of human lung pigment. i pmlsslon Spectrosoopy and X-ray Flu- titath •t a fc study • for irJ italdiE onesoence Analystsr--Zhble 4 shows the laa-, `; ftpeG jor inorganic constituents of the pigment as s• M= ; determined by emission spectrosoopy: Sili-~' -o~n dioxide is the largest component of the t; lungs inorganic group, while aluminum oxdde, iron, and phosphorus occur In ismaller-' amounts. Silica and aluminum oxides are . present in atmospheric dusts in high eonoen- trations and would be expected to be inhaled In large ts. X-ray powder diffracto- . metry of ple 44 gave a prominent a-quartz (101 line) which is in agreement Table 3r-E/emental Composition of Human Lung Pigment • Composition of Combustible Fractiont : !~ Combustible Fraction• Carbon Hydrogen Nitrogen Balance «•!^ Lung No. 'Xi Xi 'X+ : ~- C-30 79.3 54.0 8.3 9.7 28.0 ^ 7. C.47 39.5 53.6 8.9 4.6 32.9 _ C.44 64.9 76.8 4.5 2.2 16.5 •For calculations see Tabie 2. tGiculated as follows: mg carbon (as COt)/mQ combustible residue X 100: Hydrogen hnd nitrogen were caiculated from the recovery of Ht0 and NHt respectively. A part of the weight inciuded in'•8alance" represents weioht iost by InorQanic materials at 900 C. Arcli Enuiron Health-Vo1 15. Oct 1967 515 5 9 0 9 5 5 t~xami ~~ f :black ~ / ; tAelar. Upofu Oerma [ran Slack

PICMENT IN HUMAN LUNG-NEWMAN ET AL 423 20.7 35.1 so.s Table 4.-MaJor Inoryanic Components of the Noncombust7ble Residue of Human Lung Pigment ~kscHonst weight accounted for by these tour metal ozides. sCalculated as foQows: rnF of metal oddes/ms nonoomtwstible residue X 100. The .ecowry represents the with the high silica content of the samples. Other metals were ezamined spects+osoop- killy In the nonoambu~ble residue and ipere found in trace quantities. Their quanti- tive oecurrellce is fairly constant and their conoentration range Is ildicated in psrea- thedw caldtmn (0.1% to 0.6%), magneaitmi ' (0.15% to 0Z5%), sodium (0.025% , to 0.85%), titanitmm (0.05%), . manganese ;(00~1.6%), barlam (0.025% to 0005%), oop- t{p~ (0.005 to 0.0076%), >e1 (00026% y part of the car- ; to 0.0005%), lead (0.0025% to 0.005%), ~- and adjusts the ~~ (0.026% to 0 01%), samples C-30 Aned carbon to ~`std G47 eontained tin (0025e,b), sample G va' r bydro• ;44(0.25%): Traces of stnc and cerium were ,*iL 9.0%, =fola.d in-only one sample. Dae to the ttlnall ly values Famotmts of material, the amoaat~s are qtlan ient with those 1-titativeiy estimated at a ooa6deslce level of repaeations, and nposition for the lung pigmeak " uld X-ray Fla- 4 shows the ms- f the pigment as ectrosoopy. SOi-;,• xnpollent of thd :. lumtntml ozlde; ~ cur in smalkr; ' num ozldes *are : ; in high oonoailz' ced-to be inhaled ' owder diffmcW.. e a prominent is in ag>eement ~ j.:, ractkxtt I t a fattor of 2 Wyatt et a1x made a similar stlr~y of inorganic eantenbs but tbeir values for h+on may be high since the tissue they 'studied was not treated to remove •protahl, cspecially hemoglobin. Microsoopic Analsis of Ia~ 25ss<le.--~!1 atanbcr of the same black pigmented adult ltu~a used in the pnidfiaition dv~dies were eao~mtned mici~oeoepieally after stedning with a varie(y of reagents and fatbd to tihow ;~e presenoe of azy reaction of the'distx~eet b1u~c particles or of the Immediate envihoc~- EAwr Nitate Table br-Cmapadsan of R.actJats af lYpka/ Orpanic Piymuits amd the Black PipmaM of Huayae l.anp ~ Pi~inent ~ ltelasmninu '1'~i'•l Itoglobin derivatives "~ ~ Hemosiderin .~ 1% 28.0 32.9 16.5 Balance ~j Nemo(usdn Itbafiromes ~Voohrsdnn• Osrmsl autoHuoresoent tanules b ~ek Piement ot luna V11 red O; Sudan black 8. Bl.aohing Iron Test by Prussian Blue Oxidizing With Agents F.rrkyanld. Basic Upid• Fuchsla Stains fluorescence + + 0 ment that would give a due to their compo- sition (Table 5). On the basis of tllese nega- tive resultti, the pigmeat appeared to be unrelated to aqy of the me3or. dasaes pro- posed by Connor" and Pearae"; melanins, hemoglobin and ite derivatives, and lipo- chraues. Electron Mtexoeoopy.--Klectron - micro- roopy was carried out on abouit'S0 human ltmg-tisstle specimens. As indi'cated earlier in a prelimillarq aote," one can obselve the presence of elemental cart.*on In the lungs of experimental animals that have inbaled car- bon dust. The crystalline appearance of the carbon Is dharacteriatic and can be identified easily (F5R 1): In bunnan ltugs„ also, It can be distinguished readity fran silica and oth- er 3norRaric salts (F5g 2). Fbrritin also eac h0iits a characteristic patbern in the electron microscope (P56 3). Hoawer, in many of the human htnga„ we observed malty pi8- meated areas ooataining electron-opaque granules different from any of the above but sim[lar to those sieen In other tismues .vhere natural pigmeate (sa& as lipofuscin) are poesent. Tli,ese Rntnules aaggest.orRanic pig- ment (Fig 4) and the diemicxl analyses of the pigment of the.e lupp bUo point to an organic oompodtbn- . As the various ltms spedlnens were ezam- + + + • + p~ 0 0 0 0 0 . , Arch Environ Health-Vol 15• Oct 1967

42•1 !'IC,1!!:NT l\' II11t11:1N LUNC-NF.t4rA1AN ET Al, in tCt' -•_ wasfc bon w: : . and sc-. = organ`.•: _ of fer:'.-:- Fig 1.-Aggregates of carbon (C) within membrane•bound vesicles seen In the lungs of guinea t cases pig that had inhaled carbon dust sporadically for over 100 hours three months before being killed. ( from The vacuolated structures (L) contain lipid droplets (X 10,000). gorge^ •_-" SOP.:= - = ined, they were screened and sorted as to several different pigments. When the case gave :7 whether the pigment examined resembled histories of these patients, their smoking his- demo:-~:- ' - carbon, fen•ilin, ittorganic crystals, or organ- tories, and their occupations were compared ~ the }'`.=7 ic pigments. Most of the 1un's contained with the patterns of lung Iti/;ments <tbserved .lrt•/i F,nt•iru+t Ilrnlfh -lYUI l:i, nN 1!11;*

PIGMENT IN HUMAN LUNG-NEWMAN ET AL Y Fig 2.-,An alveolar septal cell from case C-44 oontaining inorganic crystailine 1. ~ ~. ~ in the electron microscope, no correlation . For example, car- ~ was found between them. bon was found i n the lungs of some smokers n and some nonsmokers; the of same was true =: organic pigment . However, a large amount. of ferritin i n the lung was found in some guinea : R cases of mitral disease as is to be expected killed. from the chronic pulmonary vascular en- ;t gorgement that occurs in these cases. Some of the pigment-containing granules the case gave a positive acid phosphatase reaction as 3king his- : demonstrated in Fig. 5. This suggests that compared the pigments may be associated with the lYsosomes and is further supported by the material, probably siliutes (5) (X 14.000). ~ evid alon the elect parti greg 425 eaoe that the black pigment sediments, g with lysosomes, which are found in mitochondrial fraction and by further ron microscopic evidence that carbon cles were seen in membran -bound ag- ates in experimental guine~ pigs. On the basis of the electron microscopic examination, three lungs with different pig- ment patterns were selected for further study and were used for the enzymatic digestions and subsequent combustion and inorganic analyses described above. Table 6 summarizes the findings. The black pigment observed at autopsy in Arch Environ Health-Vol 15, Oct 1967

f12(i PIGAfF,NT IN IIUAIAN G(1NG-NlitVAIAN ET AL Fig 3.-Section of cell from case C-47 showing aggregates of ferritin (F) (X 6.500). Table 6.-Medical History and Black Lung Pigment ih Three Men Type of Pigmcnt Case No. Age at Death OccupntUon Smoking (Pack-Years) Cause of Death . Electron Microscopy Chemical Composition 30 29 Foundry work Not known Sursery for mitral Inorganic and Mostly organic and stenosis organic some inorganic components 44 65 Heater at steel 50 Arteriosclerotic Mostly carbon. Elemental carbon. mill hcart diseasc others present inorganic and organic t -.- _- ---- componen s 47 50 0 Mrtr.rl drscasc -__-ferntin. muclr rron Watch rr-pa.r _-- Much morganir- rozrtlue. sonlc rnorL+.inir, pr;janrc cornpancrnt5 Arrh 1•;nlvirnn 1/rvihh---Vul 1:i, 00 1967

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428 PIGAIF:NT IN HUMAN LUNG-NEWMAN F,'T AG Rcuarrh r: { s researrl: 6' tutes ot Thc a Ho11is li f in the r% p Idia.^i:cn> ' data. ~ Korin 18C: b.I:o des Pir 1866. 6. L1, Pulmon ly Cens Ann lU 7. Pr: Relatioa sioru ir. Lungs. 8. \'J. Cigaret• serr.2. • 9. Rr Isolitic From I 10.1 ed Hun Physic: - h n lun e ithelial ceil treated with Gomori reagent to show distri• P •; , '.~a~ Fig 5.-Portion of a uma g i ments are present. The phos• bution of acid phosphatase. Both inorganlc (S) and organk (0) p g phatase Indicated by fine irregular precipitate (P) of kad phosphate is chiefly associated with the pigment granules of both types. (Some Is also found scattered throughout the cytoplasm, frequently the case In tissue which has been frozen and thawed before fixation as here.) (X 14.000.) solubility precludes further investigation at highly insoluble pigment, probably organic this time. in nature. Because of the extreme insolubili- 'bl to e Summary . The black pigment of the human lung has been examined chemically and with the elec- tron microscope. A method has been devel- oped for the isolation of this pigment by means of dissection followed by enzymatic digestion. It is a mixture of inorganic ma- terials, (silicates, alunlinates, and other trace metals), some elemental carbon, and a ty of the pigment, it has not been posst characterize it further chemically. Electron microscopic evi ence suggests the lung pig- ments are associated with the lysosomal particles of the cell. Comparison of the com- position of lung pigment with the medical history of the individual has failed to reveal any correlation in the group studied. rant from d b t y a s e This investigation was suppor the American \iodicai Association Rducation +nd Arch F•nuiron Heahlt-Vol 15, Oct 1967 ies of Amino 1a~ena- Amer 12. ( tion of ':. taric A ~ Ba:,a - ~ Cytol ; 14. ] m ~_A Ln Ln u, m LD t t .t

PIGMENT IN HUMAN LUNG--NEWMAN ET AL 429 datc. ~ Karin Lindquist, Mark Stephan, and Thomas Jo- seph provided technical assistance. Chester Satula. PhD, and Bill J. Wiginton, Marathon Oil Company. Englewood. Colo, did the x-ray fluooesoenee analyscs. Elemental analyses were done at the Hu/lman Laboratories, Inc., WheatridM Colo. and at the Coors SpeetraC7beaual Laboratory, Golden. Colo. Infrared absorption spectrophotometry were done on the Bectmun IR 7 spectroQhotometer and elec- tron aaiaosoopy on the Phill:ps EM-200. ReferenOCS Ceateilobalar Lestoa: Foa1 Em lersan. Aast l p y , Ann Med 6:124, 1957. ~, 7. Pi+att, P.C„ Jutabha, P., and Klusk G.A.: 'Il:e Relationshi Bet.roen Pi ment De odts and La- p € r p -. ,~Z doos in Normal and Centrilobular Emphysenatous ~tesearch Foundation, and by Public Health Service reseacch Kraot HE-0G0G7 from the National Inati- tutea of Health. : The animals used in this study were provided by $oUis Boren, MD. Roger S. Mitchell, MD, assisted• ln the evaluation of the clinical data and Coamo G. ScD in the a praisal o[ the chemical [ackenzie , p , Iu .) Pulmonary Emphy.ema: L The Genesis of the Ear- , Iamp, Amer Rev Reep Die t37:u5„ 1965. & Mitchell, R.S.; Vtnoeat, T.N.; and 191ey. G:F.: : Cgare ~~~ C>ulon~k Bronchitis, and 8mplty-. 9. ReLs, OK: StuBies of Lung Metabolism: L Isoatioa and Properties of Subo.liul.r Fiaetiona . Fevm Rabbit IAng, .1 CeU B!o[ a0:46, 1966. ` 10. Heodley, Dn. et a1: 7Le Pmperttes of Lolat- ed 8uman Qardiac Ars Pigment: L Ptrparatian and Physical Properttes, J Qeronto[ 1a:144, 1863. µ 1L F'it:patiick, M, aad Hoqielhoat. VM: 8hd- ies of Human Pulmonary Connectift Tf.wu: IL ~ ~ Amino Acid Composition of Residue FollowIng Dol- ,ably organic _; ne *tsolubili- :% !n possi'ble to ; lly. Electron the lung pig- ie lysosomal , noftheoam- t.he medical iled to reveal studied. au Po(yester pour I'Ultramiaotomie, J Ultrastruct Rea ls2M 1958,. 1b. Laft, JS: Ia:ptovetnents in Epoxy Resin ~ Met6odt~ d Biophyi Biochem Cytol 16. ReynoW $.S.: Tbe Use of Lead Citrate at High pH as an $lectroa-OpaQue Stain in Electron Micraoopy, J Ce1[ Biol 174K 1965. 17. D.aq, W Ta J[ouee Liver Lyioeomea and PhD tbeds, Ualwrodty of Leyden. 1962. Study. 1d Arrmd Foeoss Iatditate of Pathology: Manual the e~d >l: KewMoOs~a. Boo~k C~o.,, In, c~.,1980.u• 1g. $.1Lamy, L~J :~~Tk~e~ Ia~J~ro~/-~r~e~d~ Spe~e~tnao~/ C~o~mj- pfsz Motsatls.l /.~{~~~. .~.QW{.Rn and \ /~Ny {/4Y., 1960,pp 503488. =0. W~ratt, J.P., I+.cLep V.W.; aad Sweet, H.C.: The Patbomorpboof the Emphyaeuu Complex, Part L Amer Rev Reap Die S9s633, 1964. ZL SamoMdd, T4 Ocdlr, J.M.: and Keefe, JR: Tb. Fioe Stnadiam of Ltpotvadn Age Phment In the Na.mcr Sribem of Aged Mioe, .1 CeU Biol !6!l76, 1986. !2 , D D, wd Skebleq B.L.: Ensymic Aotidties o[ dn Ap Piiments: Comparative 8isbodwaioal.and B;ocLemical Studiea, Biochim Biopl" Aua 59:108„ 1965. !S. Bioom„ 031. aod 2tite.o, 8Ma Autotiuores- eant tlamoLs In 08Ls of Hnman Dermi~ II. Histo- eLembal o6ucratioo. Z zdlJor.dt 61s8(1,- 19s1. S4. Qoamoe, aL: 8toirs an IJpochromes: IV. ZLe Nataea oi te Pigments In Certain Ociana, Amer J Patb 400„ 19d8. t6. Peat.s, A+Q: Sittod:eatfs[ry, Tluoreticaf and Applted, Boston: I1tt1e„ Brown & Co., 1980. !6. Newman, JY{,; Rdn, OK: and Vatter. A.E.: x~~~ ~~. ~ t in Human !7. D.nlrabasds, L, aod Walkaborst, W.: Depo- dtfon ef b[iaMaaowls In 8uman Lues With Spe- eLi R.tat.oo6 to the Alveoiar Spaoes, Health Phys- tca 1Qs1M1,1964. PUBUC HEALTH PROQRAMS It is strange that the more pragmatic business world has produced a"ion of theories of administration while the health Ssld yleld has been neglit ible. Sinoe many otganised health services are public in nature atd auspiaes, traditional requirements of public administration have shaped orpniutional pat6eras of public health programs. The administration of such programs lus followed tLe.e.molds more blindly than crea- tively.-Yerby, Alottzo S.: Health Services AdmInlttration-Practioe in Search of a Theory, Haruard Pub Health Alunttti Bttll t4:8 (Jan) 1967. Ln ~ tn Ln kD m Arch Enuiron Health-Vol 16, Oct 1967 N : 1. Rnsen, G.: The History of Minere' Direaeet, riew York: Scbuman'a, 1943. .I Vircho.v, R: Die Pathologische Pig.nentq Vir- dwm Arch 1:379, 1847. . 8. Scheld. KF_- Ueber Exogene und Endogene giseaablaaeruosen in der Iunge, Beitr Path Anat EE?l't{. 1932. •4. Rebaamen, CA.: Die Melanose der Men- "achl&hen Brond:ialdcuesen, Virchow Arch f4%M . ism - , fI fi. Koschlalcoff: Zur Frare ueber die Entstehung :.r4 des Pigments der Luascn. Virchov Areb 35:178, ,... ism ~7 McLean, K.L.: Tlie Histology of Cieneralised 1 lareoase Digestion of Lung CaonnectFre ZS.n0es, r Amer Rev Rup Dtr 92:792, 1965. 12. Chrispe" M.J. and Vatter, A.E: Pr.scva- ~ tioa of the Ulknstlntct<tce of Plant CkUs Udn6 GIn- Add Dialdehyde as a Fixative, Nature l00:711 , `IN& I8. Bemett, HS„ and Luft, J.1L: a40al11dlne as a Basis for Bufferics Firatives, J Btophya BioeJkeia ; CytoT 6:113, 1989. 14. R,yter, A., aad KellenbetYer, E: L''includoa