—— ——— —_——-— —— ._——— _—-—I —_—I _——- _— —— —— —_——. _— —— -——- # (JD-PLO PHQTOELECTRiC EMISSION SPECTROMETRSC ANALYSIS OF THE METAL ELEMENT CONTENT IN STARCH BLOCK ELEGROPHGRE?!CALLY SEPARATE!) HUMAN S§RUM PROTEENS 'Fhosis €43 fire- Dfigtu a? M. 5. MiCfiiQAN STATE UMVERSWV Karts: Chang Nah 1954i" PHOTOWIC MSSION SPNTRG‘iEmIC ANALEIS OF THE METAL ELEMBNH'OONTENT IN STARCH BLOCK ELECTROPUQRETICALLY’SHEARATED Ell-IAN SERIH PROTEENS By Rang Chung nah A THESIS Submitted to Michigan Stat. University in partial tulltilmcnt of the requirement- tor tho degroa at MASTER OF SCIENCE Departnnnt of Biochemistry 1961; ABSTRACT PHO'IDELRCTRIC MISSION SPEC'ECMETRIC ANALYSIS OF THE METAL ELEMENT CONTENT OF S'IIARCH BLOCK ELECTROPHORETI CALLY SEPARATED HIMAN SW PROTEINS By Kong Chang mm This investigation was osrrisd out to sxtend s series of studies on ths Istal slsmsnt oontsnt of slsctrophorsticslly sspsrstsd normal human blood ssrum protsins. 'l‘hs objsctivos wsrs to adapt nsw and improvsd nsthods of establishing ths rssults on s more quantitstivs basis sad to find out about ths nstxrs snd distribution of tho Isstsl slwts associated with proteins in s biological fluid system. 1%» separation of ths sot-um protsins was sccmplishsd by «playing starch block slsctrophorssis sud subssqusnt slution with physiological sslins. Each slusts was trsstsd with FCL rsagsnt snd nonsursd spsctro- photonstricslly to obtsin dsts for s protsin distribution cum which indiostsd ths nunbsr snd qusntity of (notions sspsrstsd. All sluntss found to bs port of tho Isms protsin traction wsro combined. ooncsntrstsd. svsporstsd to drynsss. sud sshsd. Esch ssh smpls from s protsin fraction wss snalyzod in tho photoslsctric spsctro- sstsr snd thus ths mtsl slsnsnts sssocitsd with tbs slbumin. slphs-n botsc. sud gems-globulin trsctions wsrs dstsminsd. Tbs linsl dsts providsd sn sstimsts 0! tbs distribution of ths following nstsl sluonts soon: ths tour protsin trsctions soparstsd: Ca. Mg, Mn. 1's. Cu, 2n. Mo, and Al. 'l‘hs rssults indicstsd ths prsssncs of all slusnts. but not to tbs sans sxtsnt, in tho ssrus factions. VITA The author was born September 12, 1938. in Amoy. China. His secondary sducation was complstsd in 1956 st Chung Chang High School. Singapors.‘Malsysis. In 1960. hs was graduated £ron.Nanynng University. Singapors. with.s Bachelor of Scionos dogros. a. was admitted to tho Gradusts School of Michigan Stats Univsrsity in September. 1962, snd has been in sttsndancs sincs that time. ii WT The author wishes to express appreciation and gratitude to: (l) Protesoor H. A. Lillevik for guidance end counsel: (2) the Asis Foundation tor the Scholarship which ambled the suthor to schieve the participetion end sccomplishmsnt of this study: (3) Professor A. 1.. Kenworthy of the Depsrtment of Horticulture. Hichignn State University. tor the services of the photoelectric emission spectrometer: end (h) Dr. Robert 3. Roy oi Edwsrd w. Sperrow Hospital, Leasing, Michigan. for the supply at human blood senns specimens . iii TAELE OF CCNTZKTB Pace I O Il‘mcmcn 02! O O I O O O O I O O O O 0 O O C O O C O O O 1 II I Ii.:.;rP-;1;ECLKLO O O O O C O O O O I O O O O O O O O O O O O 3 A. CaICi‘xlil e e s e e e e e e e e e e e s s e e s e e e 3 E. Magnesium . . . . . . . . . . . . . . . . . . . . . u C. Iron. . . . . . . . . . . . . . . . . . . . . . . . S D. Conner. . . . . . . . . . . . . . . . . . . . . . . 6 B. Zinc. . . . . . . . . . . . . . . . . . . . . . . . 7 III. EYPSRENENTfil.HE £003. . . . . . . . . . . . . . . . . . 8 A. Apparatus . . . . . . . . . . . . . . . . . . . . . 8 1. Starch Block Electrophoresis Apneratus. . . . . 8 2. Photoelectric Emission Spectrometer . . . . . . 9 3. The Spectrophotometers. . . . . . . . . . . . . 9 B. Reagents and Solutions. . . . . . . . . . . . . . . 9 1. Starch Suspension . . . . . . . . . . . . . . . 9 2. Veronsl Buffer Solution . . . . . . . . . . . . 10 3. Folin-Ciocalteau4Lowry Reagent. . . . . . . . . 10 '. Physiolorical Saline. . . . . . . . . . . . . . 11 5. Internal Standard Solution for the Spectrograph. e s e e e e e e e e s s e s s e s 11 6. The Metal Ion Stock Standard Solution for cal ibration O O O O O C O O O O O O I O I O 11 7. Human Blood Serum Samples . . . . . . . . . . . 12 Ce Proceéure....o....o........... 12 1. Preparation of the Starch Block . . . . . . . . 12 2. Starch Electrophoresis of Normal Human B10061 germ) s e O O o s e o 0 e e O o I O O I o 13 iv 3. Elution of C .erum Proteins . . . . u. Determination of Protein Composition in Serum. . S. A31linge s e 6. Photoelectric Emission Spectrometric Determination of Metal Ions . . . IV. RESULT AND DISCUSSION . . . . . . . . . . A. Scrun Protein Distribution in the Starch Block Electrophoreticslly Separated Fractions . . B. Trace Element Composition of Whole Serum. . C. The Distribution of Metal Elements with the Proteins Fractions Obtained by Starch Electrophoresis Blood Serwn . . 1. Calcium . . 2. Magnesium . 3. Iron. . . . 4. Copper. . . 5. Zinc. . . . of Several Samples of V. SUEJLRY AIS) CONCLUSIONS . . . . . . . . . VI. IEIOGR‘APHY. . . . APP-7.33131}; Ae' e e e e e e Block Human 15 15 16 17 17 3h 39 an?! 1‘ Z S) .0... A...) W II. III. IV. LIET OF TAB Eistrib 2tion of Proteins in He in . Son 3 a teJ by Strs rch Blcck Electrrrb esis. . . . . Coupa rise n of Protein Distribution by Ptorch filo-2!: Electrophoresis of Human Elood Goran with C'tnlet' {1651 11t3. o e e o o o o o e u o o o o o o o 0 Emission 3pectrograph Readings for Metal Elements Present in Whole Human Blood serum . . . . . . . . Fetal Elenent Content in Whole Human Blood Serum SiLJE’IES. O O O O O O O O O O O O I O C O O O O O O The Distribution of “etals Asso oc at ted wi th the Protein Fractions Obtained by St .rc ch Block Electroplaoresis on Various Sampl s of human BlL~Cd.2€:rlIEJ...........o........ Emission Spectrograph Reading Values for “etal Element Content of Hu;nan Blood Serum Fractions Separated by Starch Block Electrophoresis and Corresponcing BlanL Run. . . . . . . . . . . . . . vi ‘0 C) I") ()3 28 FT Ct??? L -F 'u-\ 1. LIST OF FICURSJ Hunen Serum Protein Distribution Curve Obtained by Starch Block Electrophoresis of 1.0 Killiliter 0f A1313118d 8313:3120 o o o o o o o o o o o o o o o o o 18 Human Serum Protein Distribution Curve Obtained by Starch Block Electroghoresis of 2.0 Milliliters OEAPEIT-liedsampleoOooso...cooooocoo 19 I. INTRODUCTION A greet many proteins have become well-known in ability to form complexes with motel ions. Amony then ere the human blood serum proteins. Being in trace snounts in blood serm. the cstions. especially the transition metals. have many potential bonding orbitsls. end can interact with different electron donor groups on proteins through chelstion snd coordinate covalent bonding. One kind of cstion nsy form several types of complexes with different kinds of proteins and one protein nay fore different complexes with different cstions. Some types of bonding betwsn different functionsl groups on proteins snd different cstions have slresdy been known. but by end large. the inter- sction is not yet properly understood. Nevertheless. no setter whet kind of biological function each metal ion possesses, its binding to proteins involves highly specific sctivities in biochemical processes end is of greet significance in both physiologicsl end pathological sspscts. In spite of their fulfillment of some criteris of purity. quite s fsw'humsn serum proteins. like other serum proteins. have not been truly sepsrstsd. purified. or completely identified. It is sore con- venient to consider then.in terms of groups of proteins bssed on certain common properties which sre consequently charscterized by methods of separation such as salt-fractionation. density grsdient ultrscentrifu- gstion, chromatography, end other techniques. Electrophoresis is slso one of the principal techniques frequently used on blood serum protein seperstion end the noses of the frsctions obtained hsve been lost commonly designated ss slphs. bets. gamma. and subfrsctions thereof. It was the purpose of this study to observe both the distribution of protein-binding setsl ions end the qusntity of esch ss they occur in the fractions of norssl hissen blood seru- ss sepsrsted by stsrch block electrophoresis. Such results would serve ss reference for s study on the vsristion of these ions due to disesses or other sbnornsl physiologicsl conditions. Insseuch es the sffinity of proteins for sodium snd potsssiun ions is rether'snsll becsuse of their law‘vslencyt the distribution of these ions hes not been included in this work. -3- II . HISMICAL Several preceding reports have smarised various aspects of the historical development of the chemistry of natal proteins and netsllo— proteins in hunan blood sense. These include those submitted by LeDuc (29), Foy (13), and Long (33). In addition. the proceedings of a conference on "Biological Aspects of Metal-Binding” held at Pennsylvania State University in 1960‘ has been published with Johnson (ll) as editor. A chapter included in a text- book written by Martin (36) this year presents a sunny discussion of the role of natal ions in biological systems by giving an. introduction to for-nation constants. natal ion specificity. protein binding. and ligand field theory. Sons of the nore direct and pertinent past investigations relative to major natal ions examined in this study are reviewed as follows. A. Calcim ' Since it was suggested in nu that calciun is bound to serve proteins (M). there have been eany reports concerning this relation- ship (it. 38. 51. 1:7. 37). Copeland (8) and Carr (5) observed that nore cslcim was bound to albusin than to the globulin portion is sewn. According to LeDuc (29). two-thirds of the calciu- was distributed equally between alhnnin and the gma-globulina in huan serum. Fraud and llink (no. 1.1. 1&2) determined that 1&3 to 55 percent of all calcit- in serus was protein bound. and also concluded that 50 to SS nercent of the non-ultrafiltrable calcite: was bound to albuin. They further claimed that the Iain portion of globulin bound calciu- was in the -u- beta-globulin fraction and that only a slight amount was bound to the alpha- and gama-globulins. Previously. in 1956, Laurell. _e_1:_ 5;. (28) demonstrated that hunan serua calciun was bound to the alpha- and beta- globulins. Pathological evidences showed that the beta-24A fraction of the gonna-globulin eight be one of the proteins that bound calcius (17). It was reported by Lillevik. at _a_l_. (31) that calciua was associated with proteins in all electrophorstic fractions of lumen 3011-. A similar distribution pattern of protein seru- calciua was also supplied by Poy (13) who stated that us to 55 percent of the bound calciue was associated with albumin.‘ Long (33). however. concluded that calciun was bound only to the alpha-l- end alpha-Z—globulin fractions. Owing to differences of experimtal conditions, techniques used. and possible variation of serum protein concentration in different samples. the chances for discrepancies in the foregoing results can be more readily understood. ' ' B. Maflesium By means of an ultrafiltration method. magnesium in sea: which was protein bound was shown by Uatchorn and McCance (51) to be one- fourth of the total husan serum eagneaitn. Cantarow and Scheparts (3). in their textbook. report this value to vary froe 15 to 30 parcent. It was similarly observed by Copeland and Sundeman (8) that the amount of ngnesiua bound by elm-in was greater than that by globulins. The distribution of protein bound umsius in plasma was found by Carr (1;) to be similar to that for calcium but. on the other hand. itsipresence among all protein fractions of human serum was reported by Lillsvik, gt 3;. (31). The occurrence of magnesia in all electrophoretio frac- tions was found similarly by Foy (13). and yet Long (33) observed that it was present only in the alpha-globulin fractions as was the occu'rence of serm calcium. 0. Iron Since the 1920's. serv- (er plasma) iron has been distinguished froe hemoglobin iron (12). Later. Iarhan (1) shared that ur- iron was non-dialysable at the pl! of blood. Vahlquist (#8) in 19“ fond that both albuin and globulin could bind iron but that the main portion was in the alpha- and beta-globulin. Eolmherg and Laurell (18) elm-ted that iron was also firmly bond by serv- to form a salmsm pink colored compound. not this conpoend involved the formation of an keno-beta- glebulin complex was oufirned by Schads and Caroline (IS). losehlin (23). and Coho. _e_t_ 3;. (6). he specific iron binding beta-globalin to dial: the major part of sensm iron was bound was named ”transferrin” by Laurell (27). LeDue (29) also ascertained that sen- iron was aasociated with the beta-globulin. It was found by crosby .d Demeshek (9) and Meier (3“) that a small amount of hemoglobin normally existed in plasma or seru. This was because of haptoglebin. a protein that binds hemnglobin. 'ihus. in the alpha-z-globulin fraction where haptoglobin occurs. protein bound iron was observed. toy (ll) and Long (33) reported that iron was present in all electrophoretic fractions of the Mann rerun proteins. They also aphasined that the higher content of iron was present in the alpha- and beta-globulin fractions. 93 Connor , In 1927. Abderhalden and Holler (1) showed that copper in sans: was non-dialyzable. but on acidification. Warburg (50) found that it was released. Bisler. 35 31. (10) regarded copper as being bound to the albmin fraction after observing its migration with this sea- protein in electrophoresis. In 19118. 1101ng and Laurell (19) flared that care than 90 parent of the serum copper was firmly bound to an alpha- 2-globulin called ”oeruloplasmin" thich also possessed oxidase activity. It was determined that one mole of this protein (moleular weight '- 1Sl.000) combined with eight atoms of copper. a proportionate relation- ship was found between serum copper level and the oxidass activity shieh indicated that the sense copper level greatly depended upon the ceruloplnsmin contut (20. 21). .Lahey. g_t_ 9_l_. (25) also noted the correlation between copper level and the amount of alpha-z- and beta- globulin content. In 1953. Gubler. p; .a__l_. (15) found that. other than the firmly ceruloplasmin-bound copper. there was present a small mount of relatively lossly bound eopper vhioh probably represented this eluent in transport. By means of oral administration of Cu“ they were able to demonstrate that copper was incorporated by albumin .41 that later. through indirect exchange. it was turned over to eereloplaamin. LsDuc (29) noticed that copper migrated with the gma-globulin fraction upon paper electrophoresis. Pay (13) concluded that copper was board by all protein fractions but occurred principally in albumin and beta-globulin. Long (33) showed by means of starch block electro- phoresia that copper was associated with the albumin and alpha-globulin fractions. 8. Zinc Since the demonstration by Pauli and Schon (39) in 1921:. nine has been regarded to be protein bound. Gurd end Goodwin (16) observed the reversible combination of human norm albmin and zinc ions. l'hey provided good evidence for showing that ninc ions were bound by the imidaaole group. Vikbladh (#9) reported that ainc existed in at least two forms of linkage to protein in plaama. fibese involved a firmly bound type (30 to no percut) and a loosely bound form at physiological pH. Cohn (6) observed that nine reacted rsveraibly with different hunan plaema proteins- . Reasler. gt g}... (113) first precipitated slimmin lid game-globulin from human serm with nine ions. By means of oral ministration of Zn”. Wolff. _e_t_ 5;. (52. 53) noted that radioactive sinc largely appeared in the alhnin and alpha-globulin fractions of canine plasma. The ratio between loosely and firmly bound nine in dog plasma was similar to that seen for husan norm. and the greatest mount of nine was associated with the alpha—globulin fractions. We (29), on the other hand. found nine mainly preaent in the game—globulin. Occurrence of nine in all fractions was seen by Boy (13). and isomigration of nice with the alpha- fractions was shown by Long (33). III. EXPERIMENTAL MEEODS £2..é£22£££2£ 1. Starch Block Electrophoresis Apparatus. The instrument used was a product of the 3-0 Apparatus 00.. 538 walnut Lane. Swarthmore. Pennsylvania. Constructed of lucite. each buffer tank at the ends wen subdivided into three compartments with electrodes so that three simultaneous runs could be made. The starch block bed was placed on a 1/8 inch thick flat lucite sheet under which was the lower water cooling plate. During the performance of experiments. a sponge pressure pad was put on top of the starch block and under the upper cooling plate. Finally the whole assemblage*was clamped together with the lower cooling plate. To achieve better separation and requirement for less amount of sample. a compartment one-third the‘width of the original was constructed with the aid of the following lucite strips: No. of Stripe Direction of Us;_ Dimensions (cm.) 2 Parallel to sides h5 x 1.5 x 0.6 2 Transverse at outer 6 x 1.5 x 0.6 ends - 2 Transverse across top 7 x 1.5 x 0.6 to clamp sides together The other two-thirds of the compartment could be either used as a single compartment of twice the new size. or else as two more parallel compart~ msnts of the some new size. The 3-6 power supply unit had a range of 0-1000 volts and could supply a current varying from 0-200 milliamperes. 2. Photoelectric Emission Spectrometer (22). The emission spectrograph used was a 1.5 meter photoelectric spectrometer (commercially referred to as the "Quantograph"). with a grating containing 981 lines per mm. and manufactured by the Applied Research Laboratory. Inc.. Glendale. California. Its availability was a service of the Department of Horticulture at Michigan State University. It had a apectrographic system equipped.with automatic recording instrument. It wss designed for the analysis of the following ten elements: .Al. 3. Ca. Cu. Fe. Mg.‘un. no. P. and Zn. Cobalt was utilised as an internal standard. The excitation conditions were obtained from a "Multi-source" unit. made by the same manufacturer. During excitation. it produced a spark.discharge from 230 volts input. 900 volts output. 2 microfarads capacitance. 50 microhenries inductance. and residual resistance. The electrodes were composed of high purity graphite with the counter electrode being 1/8 inch diameter and shaped to a hemispherical point. The revolving disc-shaped electrode had a diameter of o.u92 inch and a thickness of 0.200 inch. The electrode disc revolved in the ssh solution in question and transferred it to the spark. 3. The Spectrophotometers. The models DU and B manufactured by the Beckman Instrument Co. were used for absorbency measurement at 280 and 660 mm. respectively. p, Reagents and Solutions 1. Starch Suspension. The purification of raw potato starch (Fischer. No. 8-513) was carried out by suspending 500 g. in 1 liter of 0.1 h H01 with constant -10- stirring for 15 min. The supernatant was decanted and the suspending. stirring. settling. and decanting of starch were all repeated once again with another liter of 0.1 l H01 solution. After decantation. the starch was then suspended in two voluaes of distilled water. stirred. allowed to settle. and the wash water was poured off. This washing was repeated until the starch suspension cue to a pl! value the seam as that for distilled water when Hydrion paper was used. ‘Washing once acre was carried out with glass radistilled water. The suspension was filtered on a large Buchnar funnel and the starch was allowed to dry under auction. The uni-dried starch was then washed two or three times with 50 ml. portions of veronal buffer solution (31g2,ig££g) containing a few drops of 001.. The buffer solution with 001. aided in the removal of smaller particles (fines) and impurities and helped to avoid conductivity and pH changes during electrophoresis (2.. ‘Lastly. the washed starch was suspended in an equal volume of veronal buffer and kept in the cold room until ready for use. 2. veronal Buffer Solution. The solution was prepared by dissolving 0.2n eole (no.2o g.) of anhydrous veronal (diethylbarbituric acid.'Merck and 00.). and 0.2 sole of sodium hydroxide in two liters of radistilled water. It was made to an ionic strength of 0.1 and a pa value of 0.6. 3. l'ol in-Ciocalteau-Lowry Reagent. This reagent consists of two solutions referred to ss.A and B. a. Solution A was simply the Folia-Ciocalteau reagent cal-aarcially available fro-.8ciantific Products Co». Evanston. Illinois. It is a bright yellow solution containing sodium tungstate. sodiu- molybdate. -11- lithium sulfate. phosphoric acid. and hydrochloric acid in amounts as given by Litwack (32). It was kept from light and diluted just before use. b. Solution 3 was an alkaline composition made up of the following contents: Compound Concn. gn wiv) Ratio (by vol.) Sodium carbonate a h 100 Cupric sulfate 2 1 Sodium potassium tartrate u l The sodium carbonate solution was filtered before mixing with the other two solutions. h. Physiological Saline. This was an 0.85 %»(wVV) Neal solution prepared by dissolving 17 g. of c. P. sodium chloride into distilled water until two liters of solution are obtained. 5. Internal Standard Solution for the Spectrograph. The internal standard solution (22) contained 0.0“ %.of Co (II) ion. 0.5 %»of‘Li ion. and 1.0 filo! K ion acidified with 1.8 R H01 (1500 ml. cone. not per 10 liters). ' 6. The Metal Ion Stock Standard Solution for Calibration. This solution was composed of a mixture of salts containing the ten metallic elements to be analysed. After evaporation to dryness and mixing with the internal standard solution. it was used for the calibration of the ”Quantograph." The following substances and amounts for each is as followslii=. Compound 1 ‘ Quantity (3.110 1.2 oaco, 50.06 11.30. 0.287 cm..sn,o 0.156 Peso..7u.o ~ 0.6203 znso..7u,o 1.097 ' m1..un.o 0.713 uc1..m.o 2.193 NBC]. I I1.0879 Moo. . 0.012 ugso. 3“.“ 113m. (85.0-87.0 75) 7.70s m1. 7. I Hmnan Blood Serum Smples. Pooled hunan sense samples collected from subjects were kindly supplied by Dr. R. B. Poy of the Clinical Chemical Laboratory at the Edward W. Sparrow Hospital. Lansing. Michigan. Q. Procedurg 1.. Preparation of the Starch Block. A 1/8 inch thick lucite sheet was placed over the lower cooling plate of. the apparatus. Pour lucite strips. all 1.5 cm. high. two of which are 115 cm. long and two others which are 6 cm. long. were placed in such a way that a rectangular compartment one-third as wide as the original resulted. Two other strips of 7 cm. length were placed across the top of the ends and held dom with c-clamps so as to hold the four sides of the new compartment rigid. 1An adaptation of the recommendations given by Mathis (35). The starch suspended in veronal buffer was well stirred and poured into the new compartment until the suspension became about 1 cm. high. To prevent leaking of the starch. filter paper strips were used to pack the sides of the box. .As starch settled. the supernatant‘wss gradually decanted as much as possible by the us of a medicine dropper. The. remainder of the supernatant buffer solution couldfurthsr be removed by blotting'with filter paper strips. (At the same time. while blotting. the starch block was gently pressed to exclude occluded air bubbles and thus continued until the block was dryganough to cut a slit, with a sharp spatula. When ready. the resulting starch block was approximately 0.5 cm. high. If this height was exceeded. than a greater smearing effect was noticed upon electrophoresis of serum. 2. Starch Electrophoresis of normal Human Blood Serum. a: a position 11 cm. from the cathodic end of the block. a rectangular section of damp starch 1 cm. wide and a length across of within 2 mm. of the edges was cut out. This excised wet starch was replaced by an equal volume of dried starch made from the previously described starch-buffer suspension. One ml. (or up to 2 ml.) of human blood serum was evenly streaked across the dry starch with a 1 m1. pipette. Each electrode compartment was filled with about 500 m1. of veronal buffer solution and allowed at least 30 sees. for equalisation of the levels. Both ends of the starch block were connected to the electrode compartments with 6 cm. wide whatman No. 1 filter paper strips placed above and below the starch bed. The whole block was then encased with a sheet of wax paper. followed by putting on the pressure pad and \ lllll ‘ ~1u- the upper cooling plate. The electrical circuit out attached and the current was immediately turned on. It is recommended that the ore-rent be switched on as rapidly as possible after applying theserms ample on the block as this will prevent broadening of the saople band due to the diffusion of serum. lA constant voltage or 1150 volts was observed to be the best to nintain. With a current of 20 to 2b ailliesperea (the magnitude of the current depended upon the degree of aoistening of the starch). a period of 22 to 2h»hrs.'was required for separation. Whether or not electrophoresis was accomplished could be detected visually by looking for the position of the pale yellowish brown color of the albumin none. the fastest loving protein fraction. and thus overarun could be avoided. 3. Elation of Serua.Proteins. After electrophoresis. a sharp stainless steel spatula was used to cut the starch block iate transverse slices 1 cm. apart. The two slices of starch that covered the filter paper strips on each end were discarded. Each of the other slices was placed into a consecutively numbered test tune containing 5 nt. of physiological saline solution. .After shaking for l to 2 nin.. the starch was allowed to settle. Each supernatant was poured into a centrifuge tube and rotated at 3/h full speed in the International Clinical Centrifuge for 3 to It ain. This procedure was repeated five or six times or until no additional protein could be washed out from the starch. Completeness could be detected than no further foam formation occurred in the solution during shekim. u. Determination of Protein Composition in Serum. Prom.each tube containing eluate. 1 al. was pipetted into 5 ml. of alkaline solution a of the Folia-Ciocalteaudhowry reagent (32). This mixture was kept at AO‘C in a water bath for 15 min. Into each tube was next added 0.5 ml. of freshly'diluted (ltl) Polio-Cioealteauéhowry solution A. and the mixture was allowed to stand at room tuperature for 30 win. Determination of the absorbency'of each sample was made at a wavelength of 660 mp with a neckman Model B Spectrophotometer. A graph (see Fig. l) of the absorbancy readings versus tube number was plotted and the protein fractions under each peak of the resulting curve could be identified. d.simpler eethod of detereining these amounts was to sum.up the absorbency readings of each eluate fraction and to divide this sumlby the total of abeorbancy readings in all tubes. An ultraviolet absorption determination at 280 an was also carried out on the elements by using a Bach-an Model DU Spectrophotometer to confirm the separation of the protein tractions. neweser. the areas under the peaks of the curve obtained was only proportional to protein aroaatic amino acid content and not the true proportion of all the protein fraction. 5. Aahing. a.. Ashing of the protein fractions. the eluates from the tubes of the same protein fraction were combined and the.volums of each.fraction was reduced to about 10 II. by free flame evaporation. The concentrated solutions were then transferred to separate crucibles and further evaporated in an electric air heated oven maintained at 105°C. After the contents were dry. the crucibles -15- were placed into a muffle furnace and ignited at SOO-SSO'C for 12-15 hrs. b. Aching of whole serum Rormal human blood serum was pipetted into a crucible and likewise dried and ignited as above. 6. Photoelectric Emission Spectrometric Determination of Metal Ions. a. Calibration. Calibration was carried out according ts Kenworthy (22) by using synthetic standard solutions of seven different concentrations for tea - elements. These solutions were prepared for sparking by dissolving into i g; 1.8 l RC1 the residues of the standard solution nixteres after they had been evaporated to dryness. As an internal standard. enough cobalt chloride was added to this solution to asks a 0.02% (w/v) concentration. The spectral lines used were in the second order spectre- except that for Me which was in the first order and that for sine. in the third order. b. Analysis of ashed series protein samples. the ash from mole and each fraction of serm protein was dissolved by pipetting 5 ml. of the internal standard solution into the crucible. (The volume of 5 ml. was arbitrarily chosen to be used than 0.5 g. of the protein sample was eahed. It could be varied to suit s-ples of different weight.) An aliquot of the solution was then transferred into the boat for spectroscopic analysis from which an mt was brought into ignition by the rotating disc electrode. The result was recorded automatically on a graph and the data calculated thereftoa. The results are given in Tables 11. III. IV. V. and VI. -17- IV. RESULT‘AND DISCUSSION A. Serum Protein Distribution in the Starch Block Electrophoreticallz geparated Fractions Figs. 1 and 2 show how the electrophoretic patterns appeared as obtained by the starch block technique. The solid curves were obtained by measurements using the Polin-CiocalteauALowry method for protein analysis.> The area under each peak indicates the protein content in each fraction. The dotted lines were drawn from ultraviolet absorbency readings of the eluates at 280 an wavelength. They do not indicate the true protein concentration but rather show the aromatic amino acid residue content in each fraction. They are included for supporting evidence in judging the points of separation of the serum proteins. It can be easily noticed. from the figures. that there is no sharp demarcation between peaks for the alpha-lo and alpha-Z-globulin fractions. Thus. in subsequent analysis for metal element content they were combined and are grouped as from only one traction - the alpha-globulin. Table I shows a quantitative distribution of the proteins in human blood serum as separated by starch.block electrophoresis. The protein concentrations in the fractions are reported in total absorbency units for combined eluates of the same fraction. The actual total absorbency “value corresponding to unseparated serum was obtained by using whole human blood serum of the same amount as that applied for a starch block electrophoresis run. This served as a reference base for estimating the percentage of recovery shown in Part B of Table I. -18- I: n.1,- ; tang-am. €334 no 2332:. o; - ..- e _ _ .3 admonocuaonucean Noowm £0."de am @3130 050 nouvgfiuaawn dacogm Egon ii 4 nufih ” wanton only M at c... . 3 Lb! I 4 till! III .Tlim H T a. . \/ \ J \/ -1 .-W--..- - /||\ /.1\ .. r \ lllll touuqsosqy E d 3V \' duosz WY W099 -19. .fi-V—rw ! Yuiun‘u . Md a....nonoflmohucoafi £03m meadow mm wanna-£5 ego doapgnuoan nwoacum EHLom confirm .m .u .1. a Welll‘ I ’ol'ule'lv'lt‘| ...- ~..-u.‘, -'—r. " . 7H -~ -o—h~a-_- 1., m .- b n c p a o z o p o a .odmnem doaamnd mo onoowawwawz o.m 4 ’ 4 .9“. 9x a. fir 4 con.» .__ ._ , _ \ I. . \\.\/// \\/\\\I/ \\\ H x V! .. \\ //Y \ I). | I: m \n‘, a J‘ -l .. ‘ m \b 8/. I ‘I .O ‘ow . s . I “a .I \ .I z z./. ‘a , I I I \ ... .J. s 3’ .. - ) . .... x if u, _ W a, \ M. {V _ w n, ., twfimwdv _ A » _., h a _ a . ,— i . _ _ Qai ‘I 9‘s 0 I do 09‘, «en's. we. '-D--e" ‘.‘e TABLE 1. Starch Block Blectrophoresis‘. Distribution of Proteins in Human Blood Serum Separated by Protein Fraction 1 2 3 h S 6 A. Absorbancy Units Albumin 2.h78 3.038 2.876 3.038 2.371 5.239 A1pha-globuline 1.109 0.8h5 0.553 0.62h 0.5h3 1.359 Beta-globulin. 0.865 0.812 0.627 0.518 0.386 1.296 Gamma-globulin: 2.008 1.88“ ‘ 327 1.377 0.939 2.698 T°t‘1 (‘b°"‘ 0.539 5.579 h.683 5.557 n.239 10.592 fractions) 0'13‘“‘1 §’°t'1“ 9.2u0 7.080 5.200 5.000 3.880 10.920 Content 3. Percent in Bach Fraction Albumin 38.2 36.2 52.9 Sh.7 55.9 “9.5 A1pha-globu11nl 16.5 12.8 11.8 11.2 12.8 12.8 Beta-globulins lu.6 12.3 13.h 9.3 9.1 12.2 Gamma-g1obu11nl 30.7 28.6 21.9 28.8 22.2 25.2 Recovery’ 71.0 92.9 90.1 102.9 86.9 97.0 J 1For samples 1-5. 1 al. of eluate fron each tube‘was taken. and for sample 6. 2 nl. was used. aArrived at by dilution of whole serum sample and subsequent eultiplication by dilution factor. 3See Appendix A. See Appendix.d for calculation. -21- The results of the present study are comparable to values obtained by s milar experimental procedures reported from other investigators and me thus listed in Table II. The slightly low albumin content and slightly high globulin quantity are likely due to incomplete elution of the albumin fraction from the starch block medium. The process might be made more efficient by using smaller quantities of saline to furnish more times of elution and yet give the same final volume of eluate. p. Trace Element Composition of whole Serum The emission spectrometer or "Quantograph." is structurally set up for the analysis of only the following ten elements: P. Ca. Mg. Mn. Fe. Cu. 8. In. No. and Al. Of these ten elements. only the eight metals were of interest in this study. The emission spectrograph readings of the various metal elements present in whole human serum are given in Table 111. Table IV gives in Part A the correct percentage of each element calculated from data and indicates in Part D the amount of each element in terms of mg. per 100 ml. of serum. The results show good agreement among different samples of serum. Only the percentage of iron has a greater variation among them. The cause for the high readings of alminus is unknown. According to Seibold (no). the amount of aluminum in human serum is about 17.2 gamma-percent. finally. the reading of manganese is too small to be significant. and the value for molybdenum is not too reliable because of the interference of other elements such as those due to the high concen- tration of sodium in the serum. TABLE 11. Comparison of Protein Distribution by Starch Block Electrophoresis of Human Blood Serum with Other Results. Hethgd Ref. ‘lbuin . o a o n c u o 0 610131111!" m- an a o as u o o as Alpha-l— .Alphs-Z- Beta- ‘ Gamma- Starch Block This Electrophoresis Study “6'2'55’9 11'2 l2.8 9-1’13-8 21.9-28.6 §;:::rophore.i. ( 7) u7 -71 2.7-5.8 5.1-12 u.5-15.7 11.3-2u :1:::rophoresis (13) 66.h(flV-) 3.8(‘V-2 5°“(8V°) 9.6(av.) lh.2(av.) :;f::rophora.i. (2a) 57.9(av.) 5.1(av.) 7.68(av.) 10.89(av.) 18.53(av.) iifzirophoreais (26) Sl.h-67.6 3.1-7.3 6.0-10.7 9.2-1u.6 9.8-20.2 ziazzrophoresie (30) 63.2(av.) 3.4(av.) 7.h(av.) ll.3(av.) lu.7(av.) Free Boundary Electrophoresis ( 7) 56 '62 h ’7 . 9, '11 11 '15 12 r-16 TABLE III. -23- Emission Spectrograph Readings for Metal Elements Present in Whole human Blood 8erum"'. W” - - — - - - -Sample No. - - - - - - - 312:1: ““1" 1 2 s a Ca % 0.22 0.19 0.06 0.49 Mg '7. 0.0a 0.0:. 0.11 . 0.11 Mn p.p.m. 0 0 12 12 Fe p.p.m. 69 22 69 95 Cu p.p.m. 06.7 65.8 101.0 106.2 Zn p.p.m. 26 2“ 67 60 Mo p.p.m. 1.2 1.0 2.2 2.2 A1 p.p.m. --’ --’ --’ --3 ‘Samples 1 and 2 involved the use of 5 ml. and samples 3 and h involved the use of 10 m1. of whole serum. 3Samples were ashcd at 600’0 for 17 hrs. ’Value exceeded range of spectrographic instrument. TABLE IV. Metal Element Content in Whole Hanan Blood Sens: Samples. Metal Element Units 1 2 3 a 22.2.; Ce 7. 0.02 0.02 0.02 0.02 Mg 0.00(+) 0.00“) 0.01('-) 0.01(-) Mn p.p.m. 00 - 0 0.6 A 0.6 . Fe p.p.m. 6.7 1.2 3.0 0.2 Cu p.p.e. . 0.6 6.7 5.0 5.2 Zn p.p.m. V I 2.6 2.1: 3.3 n 2.9 1 Mo p.p.m. 0.1 0.1 0.1 0.1 A1 p.p.e. ..1 «1 ..1 --1 £221.! Cs mg. 3’. 20.34 20.31; 20.314 20.314 Mg mg. 3:, 0.07 0.07 5.09 5.09 Mn mg. ‘1: 0 0 0.06 0.06 Fe mg. % 0.68 0.12 0.35 0.03 Cu mg. n 0.07 0.68 0.51 0.53 Zn mg. ‘30 0.26 0.20 0.30 0.29 Mo mg. 3’0 0.01 0.01 0.01 0.01 Al mg. 3. «J ...‘ ..1 ”1 ‘Value exceeded rsnge of spectrographic instrument. -25 C. The Distribution of Metal Elements with the Protein Fractions Obtained by StarcLBloct Electrophoresis of Several Samples of Human Blood Serum. .Table V reveals the metal element contnt present in the protein fractions of six suples of human blood serial subjected to starch block electrophoresis. At first glance they appear to be quite} random. This may be attributed to the binding of protein to the cations. As is well know. there are two kinds of protein binding combinations for cations: loosely bound and tightly bound. The cause of the inconsistent results may be due to the loosely bound portions which may not retain the smse amount of cations throughout the run of electrophoresis. Of course. artifacts (III to procedure may be a great factor in producing the noted deviation. In Table V1 is shown a comparison of emission spectrograph reading values for metal elnsnt content between serum protein fractions and their corresponding blanks. it indicates that the reagents also contribute significant interference to the results. But above all. the diminution of characteristic radiation of other ions by the excessive sodium content is probably the main consideration that causes such imprecise results. It might be partially overcome by preparing an internal standard solution containing excess amount of sodium ions to serve as a radiation buffer. Nevertheless. iron these results, an outline form or profile of the distribution pattern of each metal element in the serm protein fractions of ham blood cm: be discussed as follows: ‘0 TABLE V. -25- The Distribution of Metals Associated with the Protein Fractions Obtained by Starch Block Electrophoresis on Various Samples of Hman Blood Serum. ‘ W - - - - - -Protein Fractions - - - - - - :23. 3:23 Albumin ;- - - - -Globulins - - 4- f- - Alpha- Beta- Gama- Human Blood Serum Sample No. 1 Ca To 0.20 0.22 0.22 0.30 Mg % 0.00 0.00 0.00 0.02 Mn p.p.m. 13.2 15.3 15.3 17.3 Fe p.p.m. 98.2 141.7 811.9 39.7 Cu p.p.m. 20.8 58.0 16.8 21.9 Zn p.p.m. 322 71.2 71.2 211 Mo p.p.m. 2.7 2.8 2.8 3.3 A1 p.p.m. 6001 736‘ .J -‘ Hanan Blood Serum Bangle No, 2 Ca 93 0.27 0.19 0.16 0.30 Mg 7. 0.01 0.00 0.00 0.03 Mn p.p.e. 17.3 13.2 11.2 11.2 Pa p.p.m. 37.1 33.6 27.0 39.7 Cu p.p.m. 81.33 87.0, 10.9 ' 28.2 In p.p.m. 163 80.6 83.5 108 Mo. p.p.m. 3.1 2.7 2.3 2.9 Al p.p.m. --‘ --’ ...3 5751 Hanan Blood Mjmple No. 3 Ca ‘23 0.18 0.20 0.27 0.27 Mg Si» 0.02 0.01 0.02 0.03 Mn p.p.m. 9.7 13.2 17.3 13.2 Pa p.p.m. 28.0 25.8 36.8 31.5 Cu p.p.m. 21.0 11.3 13.8 12.7 Zn p.p.m. 168 114.2 72.3 92.6 Mo p.p.m. 2.3 2.3 2.8 2.7 A1 p.p.m. 637‘ 359 £15.151 080‘ -27- - - - - - -Protein Fractions - - - - - - :33. 332:: 11mm - - ~ - 4210150111.. - - - - - Alpha- Beta- Gamma- Human Blood_§erum Sample No. 0 Ca % 0.07 ’ 0.09 0.12 0.22 Mg - % -0.07 0.02 ‘ ' '0.02“ 0.03 Mn p.p.m. 22.0 6.1 9.7 13.2 re p.p.m. 28.6 9.2 12.7 10.3 Cu p.p.m. 20.7 5.6 9.0 11.8 2:: p.p.m. 215 ' 11.2 18.8 29.1 Mo p.p.m. 5.2 1.8 2.0 2.8 a1 p.p.m. 173 58 ‘ 112' 115 ' ' Human Blood Serum Sample No. 5 Ga 0.37 0.21 0.22 0.20 Mg 8. 0.00 0.02 0.02 0.00 Mn p.p.m. 21.3 13.3 15.2 11.2 Fe p.p.m. 36.8 20.9 30.0 15.8 Cu p.p.m. 25.9 13.3 18.3 17.8 2n p.p.m. 337 80.6 195 , 67.2 Mo p.p.m. 3.7 2.6 2.6 2.7 A1 p.p.m. 190 131 . 056‘, 302 , Human Blood Serum Sample No. 6 Ca 2 0.12 0.12 0.13 0.16 Mg 7. 0.00' 0.003 0.00' 0.01 Mn p.p.m. 10.0 10.0 8.9 8.9 Pa p.p.m. 10.8 15.5 20.6 36.9 Cu p.p.m. 11.3 9.7 7.0 12.5 Zn p.p.m. 70.5 19.7 68.6 62.6 Mo p.p.m. 1.6 1.6 1.6 1.5 A1 p.p.m. 186 206‘ 193 258‘ ’thrapolated value from curve for that particular element on spectrographic instrument. ’Values exceeding the range of spectrographic instnsaent. ’Magnesim occurs. but values becmue too small after calculation. -23- TABLE VI. Emission Spectrograph Reading‘Values for Metal Element Content of Human Blood Serum Fractions Separated by Starch Block Electrophoresis and Corresponding Blank Run‘.- W ' - - - - - -Protein Fractions - - - - - - Metal Concn. Sample Units Albumin - - - - -Globulins - . - - Alpha- Beta- Gamma— . A. 0! Serum Protein Fractions .‘ ‘ Ca % _ 10.06 0.06 0.09 0.58 Mg 31 0.02. 0.01 0.02 0.05 Mn p.p.m. 38 38 30 30 Fe p.p.m.. 56 59 78 100 Cu p.p.m. 02.0 37.0 28.0 07.6 Zn p.p.m. 283 75 260 .237 Mo p.p.m. 6.0 5.8 6.0 5.6 .11 p.p.m.. 705 9323 735 980' p. Of Lorrespondim Blank Bun Fractions . Ca 3 0.27 . 0.03 0.52 0.58 Mg 5:. 0.00 0.00 0.01 0.00 Mn p.p.m. 22 02 no 02 Fe p.p.m. 50 72 78 130 Cu p.p.m. 25.1 33.0 38.0 05.6 Zn p.15... 158 108 150 106 Mo p.p.m. 0.2 5.6 6.0 6.7 11 p.p.m. 660 922' 1116' 1135' 1The Starch Block Electrophoresis was run simultaneously in two parallel compartments: one containing applied serum sample. the other buffer only. ‘Extrapolated values from curve for that particular element on spectrographic instrument. 1. Calcium. From the result obtained and given in Table V. calciua occurs almost in even amount in each serum protein fraction. This is in contrast to many previous reports. Fraud and Flinh (01) determined that 50 to 55 percent of bound calciua was in the albuin fraction. Foy (13) gave a similar conclusion based upon paper electrophoresis: namely. that 05 to 55 percent of the bound calcius was 11.10 by albumin. Neither does it agree with LeDuc's result (29). that an equal Amaoxmt of calciua was bound by albumin and the game—globulin and that this contributed to two-thirds of the total boundcalciu. However. Fey and LeDuc substantiated the observa- tion that calciua occurred in every fraction of husan serum In Long's paper (33). calcim was reported present in only the alpha-globulin fractions in spite of the use of starch block electrophoresis as employed in this study. 2. Magnesium It can be noticed from Table V that the distribution of protein bound magnesia is similar to that for protein bound serum calciun in that it occurs in every fraction. In the subparts of Table V. the percentage of magnesium in some of the fractions is recorded as rare. This does not mean that there is an absolute absence of magnesia: in these fractions. but because the ”Quantogrsph” was calibrated to read only in percent. the small amount of magnesia present was out of this rage of measuremwt. It appears that more magnesia: is associated with the albtmin and games-globulin fractions than in the globulina. Previous studies by Lillevik 23 £1: (31) and Boy (13) also supported the finding of magnesium in all electrophoretic fractions of human blood serum. however. Long (33). using the starch block technique as in this study. presented a varying conclusion in that only alpha-globulin fractions appeared to contain magnesia. 3. Iron. r ‘ Boy (13) and Long (33) previously observed that iron was present in all fractions of buses sen- proteins after electrophoresis which is the same result found indicated in Table V; however. the contention that the nain portion of iron was found in the alpha- and beta-globulins as reported by Pay is not substantiated by the results. Instead. the albumin fraction seems to contain a slightly higher iron content than the others. High iron content is the beta-globulin fraction due to the binding of transferrin has generally’been reported in various publications (see "Historical” section). but was not observed in any of the experiesnts from,this investi- gation. 0. Copper. Protein bound copper was also found as seen in Thble V to be present in all electrophoretic fractions of human blood serum. Among the*varioua . previous reports. only the experiments of Foy (13) had a similar result. Long (33) indicated that the albumin and alpha-globulin fractions combined with copper ions. LeDuc (29) demonstrated the isomigration of copper with game-globulin fraction. Although Laurell (27) stated that lore than 90 percent of serum copper was firmly bound by ceruloplasmin (one of the alpha-z—globulin proteins). there is not very strong evidence of such a degree of complex formation in this study. -31- 5. Zinc. From the data acquired (Table V). zinc is found to be present in all serum fractions with a higher concentration mnong the albumin and game-globulin fractions. especially the former. Boy (13) also noted that nine occurred in all fractions of hum serial. whereas Long (33) reported that sine was present in the alpha-global in fractions and LcDuc (29) showed the existence of zinc with the genus-globulin. Previously. in 1950. Rassler 3g _a_1_. (03) precipitated albumin and game-globulin from lumen earns with zinc ions. -32.. V. SUMMARY AND COMUSIONS A quantitative study of the metal elements associated with the albumin, alpha-, beta-, and gamma-globulins of hm blood series was carried out by electrophoretic separation on the starch block and quantitative emission spectrographic analysis of each eluted protein fraction for the presence of Cu. Ca. Fe. Zn, bin. Al. Mg. and Mo. The experimental technique was adapted and modified to investigations on known serun proteins. and the following specific developments were noticed: A. The electrophoretic pattern shown by starch block:electrophoresis is comparable and similar to that obtained by other sons electrophoretic methods. B. The recovery of separated proteins from starch block segments was found quite efficient by elution with physiological saline solution. C. Calcium‘was found present in all the serum protein fractions and in about an even distribution. D. Magnesium was found in higher concentration with the albumin and gamma globulin fractions. 8. Iron and copper were found to be present in about equal amounts in all fractions. F. ‘Most of the sine associated with protein was found present in the albumin and gamma globulin fractions. 6. The results and experience of this study*suggest that in future researches of this nature and technique. the addition of excess sodium to the internal standard solution be included. This night then serve as a radiation buffer to minimise interference with intensity of radiation of the other natal constituents. -33- H. Another consideration suggested for future study is that the atomic absorption spectra method of analysis be adapted to determine the metal element content of the separated serum protein fractions. 7. 9. 10. 11. VI . BIBLIOGRAPHY Abderhalden. 3.. and Holler. P.. Untersuchungen uber den Gehalt des Blutsarm en Risen. Kupfsr und Meagan. z. physiol. Chem. 1.7.9;- 95—100 (1928). Barkan. (2.. Die Verteilung des leicht abspaltbaren Eisens swischen Blutkorparchen und Plasma und sein VerhaIten unter experimentellen Bedingungen. z. physiol. Chem. _l_7_l.. l9u-221 (1927). Boenendal. Zone Electrophoresis in Blocks and Columns. Edsevier Publishing Co.. New York 17. N.Y. 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The percent of each The sum of Essa readings for fraction in human = each eluate of a fraction X 130 blood serum the sun of 366° readings of all eluates Esample: The sum of the absorbency readings for all eluates in the albumin fraction of sample 1 I 2.1:.78. and the sum of absorbency readings for all eluates I 6.539 2.h78 6.539 the percent of albumin 3 X 100 I 38.2 Calculation of Percent of Recovery. An absorbency reading expressing the quantity of original blood serum was obtained by: (l) dilution with physiological saline of the same quantity of serum as used for electrophoresis to a volume equal to that of the combined eluates. and (2) multiplying the absorbency reading of this diluted solution by the number of tubes. the sum of the 5660 readings of all eluates the absorbancy reading e pressing the quantity of original blood serum The percent of recovery 8 Example: The sum of absorbency readings for all eluates in sample 2 = 6.579. and the absorbency reading expressing the quantity of the original blood serum sample ' 7.080 6.759 7.QEJ The percent of recovery 8 B. Calculation for the Metal Element Content in a Sample of Whole Blood Serum {:90 Tables IIIJ IV, and V). The emission spectrograph readings in Table III were obtained by dissolving the ash of serum sample in 5 ml. of 1.8 N HCl acid solution. If the ash of 0.5 g. of sample was used, the emission spectrograph was calibrated such that the readings would be a direct metal element content of the sample. fihen more than 0.5 ml. of serum sample was used, the readings obtained were corrected to true percentage of p.p.m. as indicated by the amount of serum sample used,, shown in the footnote of Table III. 1. Calculation of the metal Element Content of Human Blood Serum in Percent or p.p.m. Units. the emission spectro~ graph readinf weight of serum sample the percent of metal element in huaan blood serum 0.5 Egassple: Calculation of the percentage of calcium in sample 1 from Table III: the emission Spectrograph reading t 0.22 ('). Weight of 5 ml. of serum sample 3 1.017 X 5 = 5.365 g. (The weight of 1 m1. of whole serum was determined to be 1.017 g.) the percent of metal element in human blood earns 0.22 5.6s5 X 005 ‘ 0.02 ;' -41- Calculation of metal Elegant Content in Human Blood Serum in mg. per 1‘3 ml. Serum. the percent of metal element in human Llccfi scrum fetal element content I 1. x 130 x 1333 x 1,517 Z;;;L-:;)1e: The percent of Ca in Sample 1 = 0.02 0.02 Eetal element content ‘ Tfif- Y 130 Y 1300 X 1.017 ' 20.3% mg. per 183 m1. serum. balculaticn of the Fetal Element Ctntent in the Electrophoretic '12} '1 3 0 H- I o ) ‘1 4n f‘ 3"“: . a". m, ‘ 1 Huflan Flood Qerue (~ce lrnle V). ['4 G! r? 511 ll Emission spectrograph reading, w = Weight of serum sample spelled in Starch Block Electrophoresis, WE = Weight of each Berna protein fraction, 3 = weight of each metal element, N. 3 retal element content. percent of total serum, ’A \- Total volume of eluate Lcr egch fraction obtained, and '4 1! <3 H Volume of eluate in each fraction used for ashing. U = w x —— x 0.5 x m f NE W m va w 223 V s s R . E f v H «c II I t I! T“. n. . AJ . “4:31.” 131 (E! a Cu in aIEUmin of sample 1 (Table V): W I! ’39 ~‘.\-.‘U' -' [3 H LA ‘3 "1 O ‘ 0.20 X f'.1il‘.r<"'3ilh STATE UNIVERSITY LiERAF'SES II I N ; "ll‘l | 31293 03196 7270