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tt? " ...w-r^: *.*> -*- ^ ..y.aainiJaM*fid,-aftia>^ ^***'**--.w a .a<l jt*uua?u:iu:...'..na- Ai' ____ SlGPi-Y-lCS iuwl-AiCb UtjuRArUStV bapattaanr of biological tharlatry l.arvtrd ,'todlcai SiScioI at .Patar Ben l Brlghaa Hoapltal Aujuat 2, 196i Mr. Don C. Fowler, Project Manager InUrutlMul Ut-f ZtM laitirtb UritnlKtlM 297 Meet eon Avenua How York 17, Sow York Door Mr. Powlort l n fiuloalnt a copy of our raport on the project entitled "Studio* a the Biological Eatlt (or Lead Intoxication'* cover In* the flrot four aoetht i.t lt operation, ttartln* Iron April 1, >961. It you can have |oo< cult# a way In trial short tin# and I aa quit* pleated with th* projreat vo have bade. Id accord wltn your i.si'titlno I a* Iterate requ#*ttn* a ronowal of the (rant a of January 1, I94f> to IVcn.jr 31, 19o6. We latood to coolloo* tba work under to# last head Inga and In t'i itat dlroctloo Sndleatad to Our appli cation laat y*ar. bine* it# projact baa bean In oeratloo for tuch a brief lurtpj wt nay#, of cucraa, not bad an oppirtuattjr to aitand tbaaa lovaetlgac tout beyond ttia plant and hvpoiheiee prevtouily advanced uodvr tba hooding* of 1. Met uc*lv lo*y. 11. Laid and i eoe Synlhaala and III. Hata 1 lotblooala and tl.a Lrlna (.aeration of Lead, acted botii In our Initial applfcatloo and rro*ra* report. It la our lotont to do to and to Intensify th* work that t.ii .-rtt lawicnei o tuccettf ul iy. 1 do hope that you ai*d your Cocsxittea will h#r# our ant but la *t about tba progroaa of the work and w* ara looking forward to a continuation of out ple.itant ataoclatl.nl. Tbit application baa ttu> approval of tx. IU an X. klout, Profettor and tbalrwan of tba Cw partwent of blolo*lcal Qtaalairy. Th* budget for oaxt year la attachad. BlVtdkm Approvedi Slrxrtf yvurt9 C ^4. art L. Valla* Frofaaaor of Biological Cheat itry 'a V C4?-a. - '< V>.e--t~ IU*J* fc- #.{<AV# (MirMH %y>rUMnt nt Chlt(fjr v.ih, iv.,-1,; tgffipwywylJWSWS***' TWff f i Report to International Lead Zinc Research Organisation on * Project No. Ul-94 STUDY OF T1IE BIOLOGICAL BASIS FOR LEAD INTOXICATION April 1, 196S to August 1, 196S .} ,, ,`i ; August 2, 196S Submitted by: Pert L. Valla# Professor of Biological Cheatstry Biophysics Research Laboratory Department of Biological Chemistry Harvard Medical School at Peter Dent Brigham Hospital Boston, Massachusetts N 431.01 MfriftftmfcftB'failifeainii *J ' * ,-r-- '; ' i '-- . .. Progress Report-*lead Stud/ I. Introduction During the first four nonths of this project our sfforts have boon concentrstod along two ipociflc lines. First, we have explored the analytical chemistry of lead with the goal of improving methods for the determination of this natal la biological materials. The availability of suitable analytical procedures is a clear prerequisite of any significant advence In this field, basic or applied. Second, we have conducted preliminary Investigations of the interaction of lead with several biological systems of varying complexity. These were chosen with the Intent of discerning suitable experimental systems for detailed investigation of the biochemical effects of lead along the lines indicated in our draft proposal. The portlncnt studies and results which contribute to both of these goals will be described briefly. They should be considered largoly exploratory at this juncture much as s o bo of the leads already detected suy eventually servo as the basis for extendad investigations. II. Analytical Chenlstry of Lead The availability of e simple, rapid, precise, and accurate method for the determination of load in biological materials Is essential for our contemplated investigations into the biochemical effects of lead. Host mothods for the determination of traces '. )*. l*c % ,*V. .:'-H ` 1 f - * 1 > * ; a "r1- . >c. i\ ;. 2- r of lead in urine, for example, roquiro th# prior reiaoval of organic natter and concontration of load by aahing. Tho undesirable foaturos of these Mthoda aro obvioust tho tine roqulrod for aahing ia conaidorablo; aono of tho aahing procedures engender loaaes during this preparatory atop; other Mtala potentially interfere; reagents need to be purified to keep contamination at a low level. To obviate these difficulties we have investigated the suitability of atonic absorption spectroscopy for the determination of lead. Tills procedure, based on physical phenomena known for nore than 100 years, becane practical but a brief 7-I years ago through the design of appropriate hollow cathode sources by Nalsh in Australia. One of his colleagues, Killis, has applied it to the analysis of lead in urine(Nature 191. 331, 1961), In this procedure lead is extracted fron urine using anmonlua pyrrolidine dlthlocarbaaate and Rethy1 n-snyl ketone at add pit, and tho elenent dotominod by atonic absorption using the lead line at 2833 A. In this manner lead nay be concentrated by a factor of 100 or nore enabling the determination of concentrations of 0.4 ppn using SO nl samples of urine or even concentrations as low as 0.2 ppn when 100 or 200 nl sanplos of urine are used. The principles uncovered in our laboratory through the Introduction of long absorption cells, taking advantage of the - --' 'a'. I & syasswrsiwjmfmeptgi v 'rf. - --*....... t i :----, , -'H 3 Beer-Lambort law,(Fuwa and Vallee, Analytical Chemistry 3S_, 942, 1963) first aade possible the lowering of the limits of detection for many motala by orders of magnitudes. As might have been predicted on the basis of the underlying theory, these fundamentals apply also to the specific problem of lead determination and we have now succeeded, indeed, im improving the sensitivity obtainod by atomic absorption spectroscopy for this element. Our preliminary efforts have been directed toward obviating the extraction procedure while achieving the sensitivity which is required for routine analytical work. The establishment of optimal spectral conditions is basic to this procedure and, hence, we investigated the lead spectrum to uncover the ideal spectral line to employ for the measurement O of lead by means of atonic absorption. Up to the present most workers havo used tho load line at 2833 A for this purpose. Investigation of accessible lines revealed, however, that the line at 2169.99 A it Inherently more than twice as sensitive. This discovery is itself based upon our general investigation considered practical by many analysts, but which sometimes has special advantages for certain eleaients. During the examination of other hoavy metals wo have found in some Instances unlisted, very sensitive spectral lines at such relatively short wavelengths. For this roason wo have scroonod the entire spectral range from a '* 'i .A 0 t. i..j. - :-i 1 t2J70 A to 1870 Afwhilc flushing with hydrogon gas) for other possible sensitive lines. However, the absorption at 2169.99 A has, thus far, proved superior to all othors. The relative absorption of the various ultraviolet spectral lines examined is shown in Table 1. In investigating the nost suitable pathleiigth for the determination of lead we have found, thus far, that with a standard Beckman burner and hydrogen-oxygen gas a J9 centimeter pathiength Alundun cell is optiaal. Employing theso conditions a calibration curve for lead tn aqueous solution appears as shown In Fig. 1. A stellar curve is obtained In the presence of 0.1 M phosphate buffer so that under theso conditions there i no earked surpresslon of absorption clue to the presence of phosphite anio--an important consideration for the analysis of urine. In an attempt to increase sensitivity, calibration curves were prepared in the presence of varying concentrations of perchloric scid, an adjuvant we have found very helpful for the analysis of other netals. hhile at concentrations of 0,001 to 0.1 H, per chloric acid increases sensitivity slightly, concentrations aiiovc 0.1 H perchlorate give no significant enhancement of sensitivity. The loa,. calibration curve In the presence of b.OS M perchlorate Is compared with that in water in Hg. 2. I I'CJWJJ.'V'S" j.J. w t t t t :. 5 l' y?- r if- - t- St I - ft. S- We next attempted to determine the effect of urine upon the atonic absorption of lead. Urine was extracted with dlthlxone (diphenylthlocarbaxone) and carbon tetrachloride at pH 2, 4, 6, and I. A calibration curve of lead was then carried out in the presence of this extracted urine and cospared to that of water (Fig. 3). Vhen notal-extracted urine is used as a blank, as in this instance, the calibration curvo for lead is vary sinilar to that in water. However, cohered to a water blank the absorption of urine is significant. It is not inaediately apparent if this absorption is due to lead contamination of the urine saaple used or to other effects (e.g. presence of anions) but tho intrinsic urine absorption was not reaoved by extraction with dithitone. A nunber of experiments were carried out to evaluate the oxtractablllty of lead froa urine by dlthlxone since to determine the concentration of the aetal by this convenient aeans offered aany analytical advantages. Lead was added to urine and extractions were carried out over a wide range of dlthlxone concentrations and with varying pH. Using rocovery experlnonts it was detoralnod that lead was not extractable at acid pll and only partially extracted in^the pH range 4*7. Complete extraction could not be obtained in any of these prellnlnary experlnonts and thus the possibility of increasing sensitivity by concentrating load In tho urine with 1 B, r A- ft 9,'H *- dithlzone oxtraction does not appear practical on the basis of present observations. We shall next carry out the saao series of experiments employing the acnonlua pyrrolidine dithiocarbamsto reagent. Since the mixture of gases and volatilization of the sample by tho Beclean burner may not be Ideal in the case of lead ve have also investigated the suitability of utilizing specially deslgnad burners to achieve nore optimal conditions. A ring mlcroburner built in our shop, for example, pernlts fine flow regulation and alxture of three gases, adding considerable flexability to the shape and temperature control of the flame and to aspiration of the sample. The increased sensitivity for lead eeploylng such a burner is shown in Fig. 4. In this instance hydrogen was employed aa the gas feeding the ring, while air and nitrogen are supplied to the mlcroburner. Using this new burner, sensitivity is further increased about two tines suggesting that further progress along this line can be achieved. .o * The present Unit of detection of our atomic absorption method is within the range required for lead in urine by direct analysis and but a small lowering of this limit will male^the procedure suitable for wldo, routine application. In brief, these preliminary efforts to obtain adequate sensitivity for the measurement of lead have proven very 1 i 7W'i w i ^ ... encouraging end extension of these efforts should enable us to doslgn a suitable, sensitive, rapid and precise ccthod for the ftossurenent of lead in biological Materials. 111. Transferrin and conalbuain We have pointed out In our initial proposal that lead is known to interfere with lie bo synthesis at a nunber of differing onxynatlc steps. It has also been thought possible, howevor, that lead Bight Influence heco synthesis by Interfering with iron transport directly and thus preventing tho arrival of iron at synthetic sites. In view of the known natal binding propensities of seruc transferrin it was postulated that lead night bind to this protein and thus Interfere with the transport of iron. $ 'o have attempted to test this hypothesis by ssccrtslnlng if lead does indeed bind to serus transferrin in a Banner which interferes with the binding of irou. We have also carried out similar studies with tho closely related petal-binding protein, conalbunln. In order to increase the solubility of lead tho binding studies wore carried out In 1 M Trls buffer, tinder these conditions, about 1.3 egns of iron are required to saturate the iron binding sites of l Mg oi conalbunin and about 1 wgt of iron to saturate the binding sites of each eg of transferrin. The results of the competition exporieent* between lead snd iron V;UAll1,5r.^iP ' ------- .....L -w.~... - ... -.:-a .. - * . * , * * . ........... - ------- - ............... - fc`^-w^.>Yiri r^-i tw\': yj i i itr i r^frrjim"rf Aria f**--> j H-rffrfhfr- r-^aV^Ti^*1-Jfff-* -a- V-*-^y* ,,r -^.^aCg^>-^ for binding to conalbunin and tranaforrln ara shown In Pig* S and 6. Uxor con cant rat Iona of load won also tostod with similar rasults. Prom tho figures it tuy be soon that up to a 20-fold excess of lead does not interfere significantly with tho binding of iron to either conalbunin or transferrin. On the basis of these studies it does not appear likely that lead binds very readily to the iron binding site of either traasferrin or conalbunin and, thus, it seens unlikely that the competition of lead for iron-blnding in the derun plays a significant role in lead Intoxication. - IV. Hetsllothioneln Metallothioneln is a small molecular weight protein of unique chemical composition first isolated in this laboratory from hone kidney cortex(Margoshes and Vallee, J. Am. Chen. Soc, 79, 4B13, 19S7). Electrophoretlcally and ultracentrlfuglcally homogenous preparations contain x much as 5.91 cadmium, 2.21 sine, 0.21 iron, 0.11 copper and 9.31 sulfur(KXgl and Vallee, J. Biol. Cham. 235, 3460, 1960; 236, 2435, 1961). Cadmium and sine appear to conpete for binding at tho sane site and may be isomorphic in metallothioneln. Host of the sulfur is accounted for by 27 cysteine residues and there is some evidence that each atom of cadmium or tine is bound to three sulfhydryl groups The molecular weight of metallothioneln is 10,000. Bven though metallothioneln represents 1 to 21 of the -- II.IN..M.W I.* tot*1 weight of soluble prot*in in horse kidney cortex, effort* to identify it* biological function have not been successful thus far. It* physical characteristic* are consistent with a role in a wide range of potential honeostatic o chanIsas, either in detoxication, catalysis, storage or laaune phenoaeaa. The possibility that thloneln, the aetal-free protein night bind other heavy aetals, such as lead has not hitherto been Investigated. The potential significance of such binding, if denonstrated, in terns of regulation or detoxification of lead within the body Is obvious. Therefore, an Investigation of the interaction of lead with netallothloneln was undertaken. Metallothlonein was prepared according to tho nethod of EKgl and Yallee(J. Biol. Chen. 23S. 3460, 1960). The matal-free protein, thlonein, was prepared by acidification and gel flltratlon(Seph*Jei G-2S). Icnediately after separation, lead was added to an aliquot of the netal*fre* protein and the pH restored to neutrality by addition of 0.1 N sodiun hydroxide. The final concentrations enployed were: protein S x 10*4H and lead 1 x 10**14. The absorption spectra and optical rotatory dispersion of the lead netallothlonoln were then deternined. The results are shown in Pig. 7 and t. it will be noted in Pig. 7 that thloneln, the netal'^free protein, curve 1, does not absorb light at wavelengths longer than 240 a*. This it consistent with the aalno acid analyses 1 'iti *1 M " ' OiiuiSm&a i 4 . / ^xask.gavt.VniTii't tifofaifBiTTti Wi-fiV 10- i H TfTiffifrni ii i 'iV$HFtffifftffrnftfTirr :'' for thionain which demonstrates virtually complete absence of any aromatic amino acids which giva riao to tho uaual protain absorption near 280 . In contrast tha absorption spactrua of aetsllothlonein, curva 2, shows a shouldar batwaen 240 and 270 *. Diffaranca spectra(motallothlonoln minus thionain) daaonstrata tills shouldar to arlsa from an absorption band cantarad at 250 ms which compares favorably with tha known absorption bands of cadmium aarcaptidoa. Lead thionain, curve 5, shows still .s grestar absorption, with tha prasenca of a shouldar at wava* lengths longer than 300 m and a diffusa Increase In absorption at wavelengths shorter than 300 ms. This suggests that lasd binds to thionain and generates new chromophoric metal-protein ligand sites. As shown in Fig. T, tha absorption bands which appear upon binding of lead to thionain arc optically active as has been shown previously for the chrotophore characteristic of cadmium j binding to this protein(Ulmer and Yallee, Biochem. Biophys. Res. Comaun, 8, 327, 1962). Thus, tha optical rotatory dispersion is anomalous at wavelengths shorter than 350 pp due to tha presonce of multiple Cotton affects. Tha optically active load-tliloneln chronophoro Indicates that tho metal-binding site of tho protein is asymmetric and, thereby, provides a method i for further investigation of the physical chemical properties of this syltem(Ulmar and Valloo, Advances in Hniymology 7, -..,i 37, 1965). 4',`i SSWWl.f'* It would bo suspected that sulfur i tho ligand which binds load in thionein, and s o m indication that this suy bo tho cast haa boon found by studying tho intoractlon of load, as wall as othor notels, with nodel systeas. For exanple our studios of tho notal complexes of imreaptoothanol aro shown in Tablo 2. It say bo soon that at pH 8, with a 30:1 ratio of ligand to netal, tho Intoractlon of load with nercaptoethanol produces absorption bands at 250 and 310 a, with solar extinction coofflcionts at 14,000 and 3600 respectively. Tho naxlna of tho absorption bands of lead aorcaptoothanol coincides quite well with tho aldpoint of tho extrinsic Cotton effocts shown for load netallothionoin in Fig. 8. Theso investigations will font the basis of a continued study of the binding of Pb^* by various nacronolocular spcclos involved olther in storage, transport or disposal of the elerent. V. Rhodopseudosonat sphcroldos Our plans for tho investigation of tha Mechanical affects of losd Include studies of its action upon hone synthesis. As a likely nodol systen for such work, due to its ossy accessibility and known characteristics, wo hsvo examined tho suitability of Rhodopseudoaonas sphoroldes, a photosynthetic nicro-organisn which has boon successfully onployed by others in tho study of porphyrin synthosis (o.g. Gibson ot ai., Blochen. J. 8_J, 539, 1962). - i r i ? i. is * r 5* 'i / In preliminary experiments wo have grown Rhodopseudomonms spheroides, in the light, in tho presence and absence of 10**H laad. Both tha growth rata and tba extant of growth are diminished by about SOI in tha prosanca of lead. Moreover, measurement of tho chlorophyll content of tho cells grown in tha pretence of lead rovealed a SOI decrease in chlorophyll conpared to tho control, using protein as a baseline. At higher concentrations of lead, porphyrins appear to be excreted into the culture medium. These preliminary exporinents demonstrate striking effects of lead upon the total growth and chlorophyll synthesis by these microorganisms. Rhodopsoudonona&would appear to offer a unique opportunity to study tho effects of lead toxicity in a sieple systen and wo next intend in tills manner to exanino the effect of lead on the activity of various emyaes known to be required for porphyrin synthesis. VI. Conclusions During the brief duration of this project wo have under* taken investigations of the methodology for determination of lead in biological systems and havo investigated the Interactions of lead with several protolns. Xe have also shown that lead A/fects the growth and metabolic characteristlca of a simple micro-organism, Rhodopseudomonas spheroides, Which may serve as a model system for studios of porphyrin synthesis. Significant progress in these areas is already ovidont and based upon this work further efforts will proceed along the lines indicated in the Initial proposal. ........... i*s&: f> srii^A?^3vW*-. TABU I Examination of Ultravlolot Spoctrum for Load Linas By Atomic Absorption Spoctroscopy (Hydrogen-Air Plano) Linos (A) 2833 2169.99 2164 21S2 2130 ll*A) 80 ICaA) 34 33 (loss Absorption than at 2833 A) H 2093 2081 206S 2026 i 200S 1976 1962 1941 1916 1908 1900 1188 1880 1178 1872 1870 RoToTarTcVURr'175 - 5T "-rsooggR :f -; \.y _ -ISiililSl ^r ?Sv- a *- K.,, v-i* t,- > 7 a'- -- /. 5CVl*?f.v**:^t*; j,guujiiii!^.a^j4rl 'jiu-jawgipg . J1 jap^wwtw?5 IWiWW.V'W?!1 i-f'* v>'`- * ' --* . -*- ----- * J "! &' ' :- `j i 1 -j TABLB II KBTAL COMPLEXES OP MBRCAPTOBTUANOL All at pH 8, with a 50:1 ratio of UjiniiMtil CROUP METAL ION IIA VIII calciim(II) strontlua(II) barlu(Il) iron(II) cobalt(II) nlckol(II) IB coppor(I) copper(II) sllvar(I) IIB tinc(II) cadalua(II) Mrcury(II) IVA laad(II) WAVELENGTH, MAX. OR SHOULDER 266 236 280 2SS 330 264 264 275 375 226 248 <225 250 310 MOLAR EXTINCTION 1800 .. mm 3000 20000 3400 3000 11600 9600 6000 5200 5600 17400 >2400 14000 3600 * \ ^~ - ; pPPUpH^i ' ! i EFFECT OF PfRCKiORATI OH ATOMIC AbSORPrm OF IEA0 f !.- -4 .-. t o >--4 (*--* r^M4WtSWIB 0 m X .4 .6 .& 1.0 ,iC"w Cc ' 'on, -M<f/p/ml Wr. ? ta^?v *er - "',i '^ " Tngg^ary *** y,W'j>:l'Wwau.n< w ' . V^Y- COMPARISON OF ATOMIC ADSORPTION OF LEAD IN URINE vs. WATER 8a*SS8 ATOM/C A05ORPTION SPECTROSCOPY OF LEAD Pinj micro-tHjrnar v* BeeK/nanbm'ner Re/ereneat BKL 175-62 175-66 H--* BECKMAN BURWSR C45 Pft&SSMC (jbt) , UNC N* MKXGDUKHZK. aIf M' j 3.5 in t!MG OCT^Df iNS/OtyWoKWf Nx-- Ht--AIR 20 10 20 fO 14 [%-' V-VT 3.S |2 0 Lead Concentration, ppm Hflure4 a te a a llfc EFFECT OF LAD UPON DINGING OF IRON TO C0NA16UMIN Protein- tl6m$ Lead ~l07f ' -; t RrT-* ' W" iu. p S a o D ConalbUmifl *F<S O Corvolbumin +Pl>*Fa* . * *-?*-*?' *'* :.> .v,/ 5 JO 15 /ym, fe /fcference-. 6RL 256-ht> 256-m- j|)j!iyii.wvgiwpr'> J 20 25 30 Figure 5 i-ii$r*"*-'- *** ' ^,..^ir\^** TJ''? .- " TTT^ , ' 'I ''UsSpySTP' 'W&T''*-** =>"'<'' ^ -t t * -- " ^-f-- J/iV ; *v ^ 1 * ' . >V"'S>*\~i-r a rinw W.' * .<-;. *. MlaSt EFFECT OF LEAD UPOR BINDING OF IRON WTRANSFERRlN Protein- 10.95 ffl? /ir>l (ilightly furfcfcf) Lead a 207 Jr Sr Sr #o N. V Q O B Trqnsfeprio Iron O Transferrin Pb+Iroo .,, -<ip ?*% waggy-W? 10 15 /Jprn Fe M4 20 25 30 Figur* 6 *gx-: k4'p*t^#/3taKSSrS&Jnj*&vi.IT..^i*5tyf*a:?Ci.,jg^.  s '. . **r *;y 1IA11668 -2^ . ... -s*J*rtv*vf.C_ ^i^ayggagyjppgwiggjipiBea^seip^^aM^^Miawgy^^iw^ji^w^g-.^^jywts-i g^j^^^x^ywi^pwspwMjiw^ygw^ OPTICAL KOTATORY PlSPCRSiON OF TMIONE/N AMO LEAD THfOHEtN Pb-Thon9lfl '<r^T -icoo r- -- A- Tr.ioneln W l t;T- ; !. i?. j i- Si -* i. fe 5000 - * i fSor.se Kidney MetuilotHoneln ffc 240-155 ORD - Lod od<ted to tftiooein P>tc*n I*w'M Pb 0.00 M ThL pH %0 RcfcftfW. 6RL 240-255 ...J___ 500 Wore length in m/i __L 350 T$trc 8 i\ f/'.-'C**?-' ..- 2?. o ; Paraonnalt luwreh AmmUc i, full tiaa JUtiraaant, Social Sacurlty, ate.--Harvard Conpoalta Rata, 15% TachnLeiao, full tlaa Harvard Coopoalta Rata, 9 3/AX 1.950.00 5.250.00 511.08 Qifirf'si 2.500.00 Ifitpre<aw;tea. mlfe-iUf? steal Vanguard Modal 1056A Aucooatlc Ultravlolat Analyaar CVS Modal VI Fractionator with funnala and Volusatrlc'Cyltodara 3,180.00 1,200.00 itezJlm n i.oo traval 250.00 Total 328,566.88 Qyvrbaad. Harvard Rata 20% SaZIUL t82<Ll9Ul 336,260-26 1 59W iwyaw^n awi ;<^i'i5^aiaw N431.02