Document N2Yow7oOX0mqLZ7n7y5XKmj4w

-f' Agreement between the United States of America as repre sented by Consumer Protection and Environmental Health Service, PHS, DHEW and the Institute for Medical Research and Industrial Hygiene, Zagreb, Yugoslavia, Project No. 02-302-3. BIOLOGICAL SIGNIFICANCE*OF SOME METALS AS AIR "POLLUTANTS Progress report (Jan.15 - Nov.15, 1969) Principal investigatori Mirlca Ehgas, Chem.Sng., M.Sc.,Head Department for Environmental Health N36888 CONTENTS I. DETERMINATION OF GLUCOSE-6--PHOSPHATE-DEHYDROGENASE II. ALKALINE PHOSPHATASE ACTIVITY IN BLOOD SERUM III. ACTIVITY OF GLUTAMIC--PYRUVIC TRANSAMINASE AND GLUTAMIC--OXALACETIC TRANSAMINASE IN BLOOD SERUM IV. DETERMINATION OF BLOOD GLUTATHIONE V. SPECTROPHOTOMETRICAL DETERMINATION OF HEMOGLOBIN VI. DETERMINATION OF DELTA-AMINOLEVULINIC ACID IN URINE VII. DETERMINATION OF COPROPORPHYRIN IN URINE 9MR VIII. DETERMINATION OF MERCURY IN BLOOD IX. DETERMINATION OF LEAD IN AIR X. DETERMINATION OF LEAP IN BLOOD r : * DUP050311884 I DETERMINATION OP GLUCOSE-PHOSPHATE DEHYDROGENASE (G-6PD) The deficiency of glucose-6~phosphate dehydrogenase (G-6PD) activity can he measured quantitatively and qua litatively. Quantitative methods are based on reduced triphosphopyridine nucleotide (TPNH) and reduced dichloroindophenol (DGIP) light absorption measurements, while the basis for qualitative tests is the reaction of the produced TPNH either with some dyes, such as brilliant oresyl blue, dichloro-indophenol, or methylene blue, or with some basic components of erythrocytes, such as oxidized glutathione and methemoglobin. In addition, there are also tests involving production of new blood pigments or new morphologic products, as well as the test employing the TPNH fluorescence. Quantitative methods have been developed by several authors (1-1-5), They differ in the composition of the reaction mixture, temperature, and the concentration of hydrogen ions.' One group of methods is based on direct determination of the TPNH formation (1-1, -2, -3, -5) using spectrophotometry in the ultraviolet region (340 nm). The method by Ells and Kirkman (1-4) is based on the determination of enzymatic activity by means of dichloroindophenol using spectrophotometry in the visible region (620 nm). Qualitative tests are reported in the literature more frequently than quantitative ones. In dye tests (1-6-11) the enzymatic activity is the function of the dye disinte gration rate. The glutathione stability test (1-12) shows the average oonoentration of reduced gluthatione (GSH) in erythrocytes with a reduced G-6PD activity to be lower DUP05031 1-2 than erythrocytes with a normal enzymatic content. In the methemoglohin reduction test (1-12--16) the reduction is followed of the artificially produced methemoglohin hy TPNH methemoglohin reduotase in the presence of an electron carrier (methylene blue, Nile blue sulphate), The main point in methods based on the production of new blood pigments (1-17, -18) is that G-6PD deficient erythrocytes are oxidized with ascorbate more quickly than normal ones; this oxidation produces a new blood pigment which in its colour differs from the colour of the normal blood, and this allows a visual following of the reaotion. Tests with the production of new morphologic products by means of (3--(4,5-dimethylthiazolil~2)--2,5--diphenyltetrazoiium bromide (MTT) (1-19) and also fluorescent tests (1-20, -21) are of a more recent date. Four methods have been investigated* two qualitative - the method of Brewer et al. (1-13) and that of Fairbanks and Larnpe (1-9) - and two quantitative - the method of Zinkham et al. (1-3) and that of Ells and Kirkman (1-4). The methods have been developed and are being checked. However, as persons with a oongenital G-6PD deficiency are encountered very rarely, a final assessment of these methods will be possible only after employing them in a larger number of subjects, which is believed to be done by the end of 1969.. References 1-1 GlockjG.E. , McLean,?, j Further studies on the pro perties and assay of glucose--6--phosphate dehydrogenase and 6--phosphogluconate dehydrogenase of rat liver, Biochem,J,;, 55 (1953) 400. DUP050311886 1-3 Marks ,P. A.: Red cell gluoose-S-phoephate and 8-phosphogluconic dehydrogenases and nucleoside phoyphorylase, Science, 127 (1958) 1338. Zinkham,W.H., Lenhard,R.E.-, Childs,B.s A deficiency of glucose-6-phosphaie dehydrogenase aotxvity in erythro cytes from patients with favism, Bull*Johns Hopkins Hosp., 102 (1958) 169. Ells,H.A., Kirkman,H.N. s A colorimetric method for assay of erythrocytic gluoose-6-phosphate dehydro genase, Proo.Soc.Exper.Biol.Med., 106 (1961) 607, Bishop,C.: Assay of glucose-6-phosphate dehydrogenase (E.C.1.1.1.49) and 6-phosphogluoonate dehydrogenase (E.C.1.1.1.43) in red cells, J.Lab.Clin.Med., 68 (1966) 149. MQtulsky,A,G, and Campbeil-Eraut, J,M,t Population genetics of glueese-6~phosphate dehydrogenase defici ency of the red cell. In Blumberg,B.S,: Proceedings Conference on Genetic Polymorphism and Geographic Variations in Disease. New Yorks Grune & Stratton, 1961., str.159. 1--7 Bernstein,R.E.; Brilliant cresyl blue screening test for demonstrating glucose-6-phosphate dehydrogenase deficiency in red cells, Clin.Chim.Acta,, 8, (1963) 158.'. 1-8 T5nz,0., Betke,K.s Einfacher Farbtest zur Bestimmung der glucose-6--phosphatdehydrogenase in mensohliohen Erythrocyten, Klin.V/chschr., 40 (1962) 649. 1-9 Bernstein, R, E.: A rapid screening dye test for the detection of glucose-6-phosphate dehydrogenase deficiency in red cells. Nature, 94 (1962) 192. DUP050311887 1-4 IWflmflPmIt 'Ml iSfiJS igl; lips .10 Soheuch,D., Kutscher,H.: Eine einfaohe Bestimmung der Gluko se-6-Baospha tdehydrogenase als TSpfelprobe zur Verwendung als Siebtest, Ztschr.med.Laborteehnik, 3_ (1962) 22. -11 Oski,F.A., Krowney,P.M.: A simple micrometbod for the detection of erythrocyte glucose-6-phosphate dehydro genase deficiency, J.Pediat., 66. (1965) 90. -12 Dawson, J.P., Thayer ,W.W., Desforges, J.P. : Acute hemolytic anemia in the newborn infant dud to naphtha lene poisoning. Report of two oases with investigation into the mechanism of the disease, Blood, 13_ (1958) 1113. -13 Brewer,G.J., Tarlov,A.R., Alving,A.S: Methaemoglobin reduction test.- A new, simple in vitro test for identifying primaquine-sensitivity, Bull,World Health Organ,, 22 (I960) 633, -14 Bowman,J.E., Carson,P.E., Erischer,H., Kahn,M, Aimar,F.A.: A. capillary tube, nile blue methemoglobin reduction test for glucose-6-phosphate dehydrogenase deficiency. Proceedings X International Congress Blood Transfusions, Stockholm, 1964., str.592. -15 Tizianello,A., Pannaeeiulli,I,, Salvidio,E.t A simpli fied procedure for Brewer's methemoglohin reduction test, Acta haemat.', 35 (1966) 176. -16 Knutsen,C.A*, Brewer, G.J.: The raioromethemoglobin reduction test for glueose-6-phosphate dehydrogenase deficiency, Am.J.Clin., 45 (1966) 82. 1-17 RakitziSjE.T.: Test for glucose-6-phosphate dehydro genase deficiency, Lancet, 2_ (1964) 1182. m Wp "fcy DUP050311888 1-5 1-18 Jacob,H.S,, Jandl,J.H,; A simple visual screening test for G--6--PD deficiency employing ascorbate and cyanide, New Engl.J.Med., 274 (1966) 1162* 1-19 Fairbanks,V.F., Lampe, L. T.: A tetrazolium-linked oytochemical method for estimation of glucose-6phosphate dehydrogenase activity in individual erythro oytes: Applications in the study of heterozygoies for glucose-6-phosphate dehydrogenase deficiency. Blood, 31 (1968) 589. 1-20 Beutler,E.: A series of new screening procedures for pyruvate kinase deficiency, glucose-6-phosphate dehydrogenase deficiency, and glutathione reductase deficiency, Blood, 28. (1966) 553. X--21 Beutler,E.', Mitchell,M*-: Special modifications of the fluorescent screening method for gluoose-6-phosphate deyhdrogenase deficiency, Blood, 32. (1968) 816, at JilifS DUP050311889 II ALKALINE PHOSPHATASE ACTIVITY IN BLOOD SERUM For the estimation of the aotivity of alkaline phos phatase the method hy King and Armstrong was selected (n~l) which is Based on the measurement of hydrolysis of disodium phenyl phosphate By the enzyme at 37C yielding equivalent amounts of phenol and phosphate. After the precipitation of proteins with the Folin-Ciocalteu's reagent the liberated phenol is determined by speotrophotometrio mea surement of the optioal density (620 nm) of the blue oolour formed on addition of sodium carbonate. KingArms trong 's unit of alkaline phosphatase aotivity is defined as the amount of enzyme which liberates 1 mg of phenol from 0.005 M disodium phenyl phosphate within 15 minutes at 37C in a oarbonate-bioarbonate buffer pH 10.' The method was calibrated with @ri of pure phenol solutions of different concentrations. The regression equation, obtained by the method of least squares, reads as follows! mgs of phenol = 34.97 x optioal density - ' Milligrams of phenol are equal to the number of K.A. units. In Table II-l the results are presented of the esti mation of alkaline phosphatase aotivity in 32 '`normal" serum samples.' The results obtained are within the normal range proposed in the literature (II-l). ||| liSiiij DUP050311890 II--2 Table II--1 \ Number of Alkaline phospha sample tase activity (E.A. units) 1 3.74 2 4.89 3 3.95 4 3.53 5 3.25 6 6.78 7 2.34 8 4.06 9 6.05 10 7.90 11 5.17 12 4.30 13 5.91 14 6.29 15 2.76 16 3.74 Number of Alkaline phospha sample tase activity (K.A. units) 17 4,76 18 4.48 19 4.41 20 6.08 21 , 5.24 22 6,12 23 6.54 24 7.27 25 5.35 26 6.26 27 6.85 28 5.73 29 6.22 30 5.14 31 7.34 32 6.08 Amelung et al. (II-2) indicated that the activities of most frequently used enzymes in clinical biochemistry are sufficiently stable at room temperature only up to 6 hours following the collection of blood samples. As the majority of blood samples to be analysed in the oourse of the present project are to be collected far from the labora tory and will have to be transported to the laboratory and analysed with a delay} the stability of the enzyme at va rious temperatures and through various storage periods is being studied. m IpM DUP050311891 II-3 r.'igE 3Ti References II-l King,E.J., Armstrong,A.R*s Can.Med.Assoc.J., 31 (1934) 376. 11--2 l * ;3-is Sr:-fWh- Arne lung tD et al.s Deut.Med.Wsohr., 91 (1966) 851, wm jS|l ww > _ Stlliki Mam ,,,ss DUP050311892 Ill ACTIVITY OF GLUTAMIC-PYRUVIC TRANSAMINASE AND GLUTAMIC--OXALACETIC TRANSAMINASE IN BLOOD SERUM The simplified colorimetric method described by Reitman and Franhel (III-l) was chosen for the assay of serum glutamic-pyruvic transaminase (SGPT) and serum glutamic-oxalacetic transaminase (SCOT). The estimation of SGPT activity is based on the liberation of pyruvate from a substrate containing c<-keto-glutario aoid and dl-aspartio aoid* The formed pyruvate Is subsequently determined oolorimetrically in the form of pyruvate hydrazone. The SGOT activity is estimated by determining the change of substrate containing cK-keto-glutaric aoid and dl-alanine yielding oxalacetate which is subsequently -M' partially transformed into pyruvate. The hydrazones of the mixture of oxalaoetate and pyruvate are determined colorimetrioally. In Fig.III-l the obtained calibration curves for the estimation of SGPT and SGOT are presented. In table III--1 and III--2 the results are presented of the estimation of SGPT and SGOT resp., in 32 "normal" human serum samples. The results are within the normal Wmm range as cited in the literatures (III-2), DUP050311 893 DUP050311 894 400 Fig. iu/4 125 450 umrs of enzyme Ill--2 Table III-l Number of SGPT activity samples (Karmen units) 1 9.0 2 8.0 3 2.5 4 2.6 5 3.0 6 6.0 7 2.0 8 2.5 9 3.5 10 3.0 11 2.0 12 2.5 13 1.5 14 8.0 15 6.0 16 1.0 ' Number of . SGPT activity sample (Karmen units) 17 5.0 18 9.5 19 5.0 20 8.5 21 . 3.0 22 5.5 23 4.0 24 5.0 25 5.5 26 13.5 27 4.5 28 spoiled 29 3.0 30 3.0 31 1.5 32 3.5 .Amelung et al, (III-3) have emphasised the greater instability of transaminases than that of alkaline phos phatase. For the same reason as mentioned under "alkaline phosphatase" the stability of SGPT and SGOT through various storage periods at various temperatures is being studied. DUP050311895 Table III-2 Number of SGOT activity samples (Karmen units) 1 11.5 2 11.5 3 10.0 4 8.0 5 9*0 6 9.5 7 8.0 8 10.0 9 8.5 10 9.0 11 9.0 12 8.0 13 T.O 14 10.0 15 11.0 16 8.5 Number of sample 17 18 19 20 21 22 23 24 25 26 . 27 28 29 30 31 32 SGOT activity (Karmen units) 7.0 8.0 8.0 12.5 11.0 * 9.5 11.0 7.0 13.5 12.0 12.0 11.5 11.0 9.5 8.5 12.0 References III-l Reitman,S,, Frankel,S.: .fim.J.Clin.Pathol., 28 (1957) 56* III--2 Chinski.M. et al.: J.Lab.Clin.Med., 47 (1956) 108. Ill--3 Amelung,D. et al.s Deut.Med.V/schr., 91. (1966) 851. ll DUP050311896 IV DETERMINATION OF BLOOD GLUTATHIONE Methods Tor the glutathione determination take advantage of two glutathione properties: (1) that it activates the glyoxalase enzyme which transforms methylglyoxal in to lactic acid, and (2) that it contains sulfhydril groups which are very reactive. In our inve stigations we decided in favour of the methods basSd on the oxidation of sulfhydril groups, of which photometric methods either with nitroprusside (IV-1, --2) or*the Ellman (5,5-ditiobis-2-nitrobenzoio acid) (DTNB) reagent (IV--3) are best known. Methods with nitroprusside are not suitable for routine investigations securing no colour and reagent stability and being extremely sensitive to temperature. There are no such shortcomings with the Beutler method with DTNB (IV-3) developed on the basis of Ellman's determination of sulfhydril groups (IV-4), and for this reason this method has been selected as the most suitable. The maximum absorption for reduced DTNB in the visible part of the spectrum has proved to be at 412 nm (Fig.IV-1). The experimental points for various concen trations of glutathione in water and the same standard added to 0.2 ml blood are shown in Fig.IV-2. The results of the analysis of glutathione in blood and water are represented with a single regression line sinoe the difference in the slope and in the variance of the two individual regression lines were not statistically significant (see the table). DUP050311897 BMfc ffT m- - _i_ TT I i 441i \ V \, 1A i t 00 4 J 11 I ' '1 .i i !i i 11 !^ _ _L 41 - i 44i_.L.. _LL i &Ti.=;nT?P'PTOM nnRW.Q wnp : -' - ij _ULU iAxfcL lO JN JS EEDuCED TP JUJUHi3 `I .i t ; M! .il1 1 i nm r rr flolTd r \ | I' T i 11 I1 f .1...j..1j.... ~~1M :i--l1 1t U :> i i i J . .j "f ( Ti tz\ OA it i w i1 11* ft 7 0 i *1 ir - L 4-U \! TO U ' l\ _L -----1. * 1J70 r L L A J I iLL ll 1! .JL J1 1 1 _ _* ll ' K. \ 1 1 1 - li -- 1 1 1 1 u11... \I 1L f - .i 1! I LiM* -il! * n r in 71 4 PlaTT I J fsffS ii it ....LL i l * --r i 1 rt ii 1 < iU^TUTWtmA\Anir.rUrTAX'i31 Tlii j rn mi 1 ,. i 'I % E miis } ;JJ-. .J ..... PE Et ML 1 _LU_i_il i : TTLET i :r - i --i1 , Fig.IV-1 DUP050311 898 <oD o 0S3 teO & <ora DUP050311899 blood water b s2 s sb Ab t *0.05(V =20) 0.01614 0.1665 0.733 0.651 0.0271 0.0255 0.000236 0.000222 0.000513 1. 6 2. 086 F 1.12 P > 0.05 4 fc The method has been ekeohed on 10 blood samples of healthy subjects.; The glutathione oonoentrations ranged from 65 to 79 mg per 100 ml of blood. Phrther investiga tions on the normal glutathione concentration are in progress.' References IV-1 Grunert,R.R. and Phillips ,P.H.: A Modification of the Nitroprusside Method of Analysis for Glutathione t Arch.Biochem*, 30 (1951) 217. IV--2 Beutler ,E.': The Glutathione Instability of DrugSensitive Red Cells. A New Method for the in Vitro Detection of Drug Sensitivity, J.Lab.Clin.Med., 49 (1957) 84. IV--3 Beutler,E., Duron,0. and Mikus Kelly,B.: Improved Method for the Determination of Blood Glutathione, J.Lab.Clin.Med., 61 (1963) 882. IV-4 Ellman,G.`L.: Tissue Sulfhydryl Groups, Arch.Biochem. Biophys., 82 (1959) 70. DUP050311900 SPECTROPHOTOMETRICAL DETERMINATION OF HEMOGLOBIN (Hb) To determine hemoglobin (Hb) speoirophotometrically, the oyanmethemoglobin method has been used. Its principle is that by means of an oxidant (potassium ferrieyanide) and cyanide the blood hemoglobin is transformed into oyanmethemoglobin (hemiglobinoyanide, HiCN) which has a wide absorption band with the maximum absorption at 540 nm. By measuring the intensity of the coloured complex and taking into account the dilution value, it is possible, using the standard with the known Hb concentration, to determine Hb in the sample examined. The International Committee for Standardization in Haematology (ICSH) (V-l) has strictly defined conditions of the analysis and recommended, as a universal standard, the solution containing 52.2 mg HiCN in 100 ml and cor responding to the concentration of 14.36 g Hb. Such standards are produced in Europe by the Institut voor de Volksgezondheid, Utrecht, the Netherlands, and our preliminary investigations have been conducted by means of such a standard which contained 59.0 mg HiCN and was equivalent to 14,8 g Hb. The extinction coefficient at the wave length of 540 nm was found to be . * 44.6, and the factor for further calculations was determined as f = 36.7. Both values proved to be in agreement with the characteristics of the standards determined by the Rijks Institut. Analysing the stability of the oyanmethemoglobin complex it has been evidenced that in the interval of 0.5 to 24 hours, at 4C, the colour of hemiglobinoyanide DUP050311901 practically remains unchanged. Several blood samples were analysed simultaneously, and the reproducibility of the results proved satisfactory* Investigations of normal values in a larger popula tion are in progress* Reference & V--1 International Committee for Standardization in Haematology, Brit.J.Eaematol*, L3 (Suppl.) (1967) 71. DUP050311902 VI DETERMINATION OF DELTA--AMINOLEVULINIC ACID (ALA) IN URINE According to literature data, the method of MauzerallGraniok (VI-1) is used most frequently for the determination of delta-aminolevulinic aoid (ALA) in oases of lead exposure and lead intoxication. The method is very sensi tive hut time-consuming and therefore not always suitable for routine investigations in large populations. The method employing alkaline picrate after Schuster (VI-2)*, though quick, is not suitable either, because it cannot separate porfobilinogen which interferes with the reaction of ALA with picric acid, Porfobilinogen also interefers in the reaotion with p--dimethy1-amino-benzaldehid reagent (DMAB) but the method of Mauzerall-Granick separates it by means of the anion-exchange resin Dovex-2, However, according to some authors (VI-3, -4, -5), porfobilinogen is rarely excreted in increased quantities in lead poisoning. On the basis of these observations Williams and Few (VI-6) have concluded that the porfobilinogen determination is superfluous if ALA is used as a screening test in workers exposed to lead. They have modified the Mauzerall-Graniok method by neglecting the porfobilinogen determination and also ohanging the conditions of ALA elution and ALA condensation with aeetil-aoetone. The method was tested, against the original method, in a group of exposed workers and the results proved very good (the correlation coefficient r = 0,99). Grabecki et al. (VI-7), neglected both the porfobilinogen and the urea removal shortening in this way the procedure time considerably. By a combined method of Mehani (VI-8) ALA is separated from porfobilinogen by the Mauzerall-Graniok DUP05031 1903 VI-2 method and further analysed hy the Schuster method. In this way, in the author's opinion, the advantages of both methods are preserved: there is no interference of porfobilinogen with the reaction with picric aoid and the speed of the procedure is considerably increased. From the methods described the method of Mauzerall-- Granick (VI--1) and that of Mehani (VI-8) have been investigated so far. The modification after Williams and Few (VI--6) of the Mauzerall-Granick method, has been adopted, that is the first 3 ml 0,5N NA acetate passed through the Dowex-50 column during the ALA elution are not discarded because this eluate has proved to contain some ALA as well. The elution is conducted by first adding 4,5 ml and then 5,0 ml 0.5N NA acetate, and then the combined eluates col lected in one container. Measurements of the absorption spectra in the 700-400 nm range for the ALA and DMAB product have shown that for the Beckmann DB--G- spectrophotometer the maximum absorption is at 555 nm (Fig.VI-1). The standardization of the method has been oarried out with the known oonoentrations of ALA x HC1 in water and the same concentrations added to the urine. For the 0-100 pg/ml range, and the optical path of 0,5 om, the curve has proved to follow the Beer-Lambert law (Fig.VI-2). The Mehani method (VI-8) has been slightly modified. The saturated piorio aoid solution, originally suggested by the author, has been replaced by 0.3M solution. The optimal amount of picric acid of this concentration for the reaction with ALA is 0,3 ml. As regards the amount and DUP050311 904 DUP050311905 DUP050311906 VI-3 concentration of sodium hydroxide and hydrochloric acid no changes have been introduced except that the shaking time is strictly defined, amounting 5 minutes after addition of sodium hydroxide and 5 minutes after addition of hydrochloric acid. Measurements of the absorption spectrum for the 700-250 nm wave lenght range have shown that for the absorption measurement of the ALA-Na piorate product the wave lenght range in the vicinity of 500 nm is far more suitable than that in the vicinity of 450 nm (Pig.VI--3) as suggested by the author. Thus, on the basis of a series of analyses, the wave lenght of 495 nm has been selected as the most suitable. The method is standardized with the known concen trations of ALA. x HC1 in water and the same concentrations in the urine. The standard curves at the 495 nm wav lenght and 0,5 om optical path are shown along with the standard curves for the Mauzerall-Graniek method (Pig.VI-1). These curves, too, follow the Beer-Lambert law for the 0-100 ^ig/ml concentration. The effect of creatinine has been analysed separately. In addition to reacting with ALA, alkaline pierate also reacts with oreatinine (VI-9) normally contained in the urine, while the added hydrochloric aoid is supposed to remove this interference. By using the known creatinine concentration (100 ^Jig/ml) it has been proved that under the conditions described, the effect of oreatinine is completely eliminated. By summing up the results obtained in the application of Mauzerall-Graniek and Mehani methods, it may be concluded DUP050311907 DUP050311908 VI-4 that both Methods eaa he applied ior the determination of ALA concentrations in the urine* However, as regards sensitivity, the Mauzerall-Graniok method appears pre ferable* Further investigations should clarify if the modification after Williams-Few, i.e. omission of porfobilinogen removal, could be used, because it would simplify and speed, up the procedure and make it more suitable for the examination of a larger number of subjects* References VI-1 Mauzerall,0*, Granick,S.: The occurrence and deter mination of o^-aminolaevulic acid and porfobilinogen in urine, J.Biol.Chem., 232 (1958) 1141. VI-2 Schuster,L; `Ehe determination of f^-aminolaevulio acid, Bioohem.J.Lond., 64 (1956) 101. VI-3 Griggs,R.C., Harris,J.W.; Erythrocyte survival and heme synthesis in lead poisoning, Clin.Res*, 2. (1958) 188. VI-4 Haeger-Aronsen,B. ; Studies on urinary excretion of -amino-laevulio acid and other haem precursos in lead workers and lead-intoeicated rabbits, Soand.J. Clin.Lab.Invest., 12. (1960), Suppl.47. VI-5 Kretzer ,A.J.de, Waldron,H.A.: Urinary delta aminolaevulinlo acid and porphobilinogen in lead-exposed Workers, Brit.J.Industr.Med., 20 (1963) 35. VI-6 Williams,M.K., Few,J.D.: A simplified procedure for the determination of urinary i/'-aminolaevulinlo acid, Brit.J.Ind.Med., 24 (1969) 294. DUP050311909 VI-5 VI-7 Grabechi, J., Haduch,T., Urbanowioz,H. t Die einfaehen. Bestimmungs-methoden der cT-AminolSvulinsSure 1m Harn, Int.Arch.Gewerbepath. Gewerbehyg., 23 (1967) 226. VI-8 Mehani,S.;: A rapid method for the determination of delta amino-la evulinic acid in urine, Brit.J. Industr.Med., 21 (1964) 78. VI-9 Jaffe,M. s Ueber den Niedersohlag, welohen PiKrinsSure in normalem Harn erzeugt und Bber eine neue Reaction des Kreatinins, Hoppe-Scylers Z.physlol.Chein., 10 (1886) 391; DUP050311910 VII DETERMINATION OP COPROPORFHYRIN IN URINE A modification of the fluorimetrio method proposed 1 1 J; | by Schwartz et al, (VII-I) has been used in our laborato ries for the determination of total urinary eoproporphyrine for a number of years* In the original method the urine sample is acidified with buffered acetic acid, the copro porphyrin is extracted with ethyl acetate, the extract washed with sodium acetate, the ooproporphyrin precursors oxidized with iodine, and the ooproporphyrin is finally extracted by several successive portions of hydrochlorid acid. The fluorescence intensity of the last extract is measured and the ooproporphyrin concentration read from a calibration curve. We have shown that chlorine ions in the last extract exhibit a quenching effect on the fluorescence of copro porphyria lowering the sensitivity of the method and that this can be obviated by subtituting sulphuric for hydro chloric acid (VII--2), The quenching effeot is of practical significance only at very high ooproporphyrin concentra tions, as can be seen from Pig,VIl-l where two fluorimetrio calibration ourves with coproporphyria in 1,5 N hydrochloric acid and two in 10# sulphuric acid are presented. They show the dependence of relative fluorescence intensity upon the ooproporphyrin concentration. Two ourves were obtained using a home-made fluorimeter with the exciting radiation of mainly 365 nm, and two ourves using a Parrand fluorime ter with the exciting radiation of mainly 405 nm. In addition to the afore mentioned quenching effeot of chlorine ions on the ooproporphyrin fluorescence, the main reason for which we have switched over to Idle 5 ? DUP050311911 1> r << l'Y "M';-' '' ' i\^;. , W ' 'V U * ' ' - ; S\' 1 ; v. !;, ' . ' - >t .-.l '. vVy. V V. M: g i> o <J <T nn i ii !a- -" jug/ml final solution COPROPORPHYRIW COUCEWTRATIOkl > o o> u. 00 Is to eo at oo DUP050311912 IV sulphuric aoid final extraction in the urine analysis for coproporphyrin is the very strong corrosive effect i '! ' of hydrogen chloride which evaporates from the 1.5 N ('V,l hydroohlorio acid in the fluorimeter in spite of covered *Mj# Cuvettes. ^ i ' ** , A*In the present project urinary analysis of ooproporphy-- .^Vv'lii' rin I and III isomers is planned to be performed in addition to the determination of total urinary ooproporphy- ;'x/iV\VU'-' i . a part of sufcjeots under investigation. We have chosen the thin-layer chromatographio method described by Jensen (VII-3), and P.Kosicplo and I.Toivonen (VII-4) for the analysis of ooproporphyrin isomers. In this method I.u the extraction of ooproporphyrin is performed with ether v-i. instead of ethyl aoetate and the final extraction with hydro* 1 ' ' chloric acid. Besides, 60 -- 80 ml of urine is needed for the isomer analysis Instead of 5 ml only needed for the determination of total ooproporphyrin* As we want to use the same urine sample for both the determination of total urinary ooproporphyrin and the analysis of isomers, and in order to avoid totally separate analyses for the two ft. v: .v purposes, we are trying to modify the methods. Our experi- ments conducted so far have shown that the substitution of ether for ethyl aoetate lowers the sensitivity of the method for the determination of total ooproporphyrin. !; > Whether this effect is due to the positive Influence of 1 ethyl acetate on the fluoresoence intensity of coproporphy-- &ftp V rin or to reduced extraotibility of ooproporphyrin by ether, #1 is not yet clarified.1 The experiments are being made to ohech whether sulphuric aoid used for the final extraction in our method would interfere with the isomer analysis.* 4 f1p- DUP050311913 VT1-3 Briksea has suggested (VII-4) that the oxidation of ooproporphyrin precursors with iodine might change the isomer distribution* We have therefore checked the role * of iodine in the coproporphyrin analysis and found that iodine, if applied as suggested in the'original procedure of Schwartz et al. (VII-1), lowers the results of total ooproporphyrin determination. The iodine concentration in the reaction solution ought to be reduced or iodine possibly ommitted if further experiments show that the precursors are quantitatively transformed into ooproporphy rin on standing without addition of an oxidant (which assumption is based on some experimental evidenoe produced in preliminary testing), and particularly if they show at the same time that the oxidant does change the distri bution of isomers. If the isomer analysis is not to be included into the project, the analysis of total urinary ooproporphyrin will be made by our present modification of the method proposed by Sohwartz et al,' further modified as to the oxidation of precursors.- References VII-1 Schwartz,S., Zieve,L. and Watson,C.J.x J.Lab.ciin.* Med. , 37 (1951) 843 VII--2 Valid, F., Skurid,Z. and Noweir ,M.H.x J.Egypt.Pub.Health,Assoo., 41 (1966) 289, VII--3 Jensen, J. x J.Gbromatog., 10 (1963) 236. VII-4 Koskelo,P.. and Toivonen, J,: Soaadinav. J.Clin, and Lab. Investigation, 18. (1966) 543. VII--5 Eriksen,L.x Cit.after Nr. 4. DUP050311914 VIII DETERMINATION OP MERCURY IN BLOOD The photometric method proposed by Jacobs and co workers was ohosen for the determination of mercury in blood (VIII-1, -2), The method consists of an incomplete digestion of the biological sample, subsequent extraction of mercury with dithizone, decomposition of the dithizone complex by heating to liberate the mercury in the form of vapour, and final determination of mercury by ultraviolet photometry in gaseous phase* The combustion-ultraviolet photometric equipment consists of a heating chamber connected to a U-tube con taining non-absorbent cotton connected in turn to a cold trap, a rotameter, an additional cold trap, a modified Beckman mercury vapour meter Model 23, and a vacuum pump* The mercury vapour meter is modified by replacing the grille with a cylindrical optical oell with removable silica end windows and inlet and outlet ports for the air stream drawn by the vacuum pump. The heating ohamber consists of a Bunsen burner mounted in a Niohrome wire cage in opposite sides of which two openings are out for a Fyrex ignition tube. The igni tion tube passes through a wire cylinder fitted between two openings of the heating chamber* The calibration curve for the estimation of mercury from the readings on the Beckman mercury meter is presented in Fig.VIII-1. The sensitivity of the method obtained was 3 x 10"9 grams of mercury per milliliter of blood* In table VIII--l the results are presented of mercury determination in 32 blood samples taken from subjeots with no known exposure to mercury* DUP050311915 STANDARD CURVE FOR MERCURY ESTIMATION f j !i DUP050311916 ftgp/1 Maximum reading -xltf VII1-2 Table YIII-1 Number of Mercury sample ^ig/100 ml 1 0.60 2 0.30 3 < 0.30 4 < 0.30 5 < 0.30 6 < 0.30 7 0.30 8 < 0.30 9 0.30 10 < 0,30 11 0.60 12 0.75 13 2.10 14 0.60 15 0,90 16 1.20 Number of sample 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Mercury ^pg/100 ml <0.30 1.65 0.90 1.65 1.80 2."70 6.00 0.90 2.40 5.10 3.60 3,78 5.40 5.10 6.00 6.00 As can be seen from the table, mercury content of 7 samples (about 22%) was below the analytical sensitivity of the method (0.3 /lg per 100 ml). In order to avoid "zero" results (or rather results indicating the concentrations below 0.3 ^tg/100 ml) in analyses of "normal** human blood samples two steps have been tahent 1.' An attempt has been made to avoid the measurement of the ultraviolet radiation absorption by mercury vapour in a dynamic system i.e. in the ourrent of air, as is DUP050311917 VIII-3 being proposed in th original method* The optical cell proposed to be used in the mercury ultraviolet photometer has been made tight so that vacuum oan be created inside the cell* By means of this vacuum the total quantity of meroury is drawn into the cell and the absorbance of mercurymeasured in the statical system. The results obtained thus far do not yet permit a definite conclusion as to the applicability of the modi fication* 2. The method recently suggested by Magos and Cernik (VIII-3) for rapid estimation of meroury in undigested biological samples is being introduced and checked* The method consists of liberating mercury from suifhydryl bonds by means of stannous chloride in the presenoe of oystein, and subsequent measurement of mercury vapour In the same ultraviolet photometer. The checking of the method will be completed within two months. References VIII--1 JacobstM.B. et al;j Aa. Ind.Hyg.Assoc. J, , 21^ (1960) 475. VIII--2 Jacobs ,M.B. et al.s Ibid., 22 (1961) 276. VIII-3 Magos,L., Gernik,A.A. * Brit.J.Ind.Med., 26 (1969) .144. DUP050311918 IX DETERMINATION OF DEAD IN AIR A ring-oven method (IX-l) for the determination of lead as chromate, developed earlier (XX-2), was applied for the analysis of low volume samples of air-borne particulates and verified against atomic absorption speotrophotometrie (AAS) determination of lead in parallel high volume (HV) samples. Two wet ashing procedures for the preparation of HV samples for analysis were also compared* An attempt was made to compare just the two analytical methods by analysing the aliquots of the same HV samples by both methods, thus eliminating the influ ence of sampling efficiency on the results. The relative change in the flow rate during the sampling period was recorded for both sampling sistems and compared* The retention of lead in the low volume sampling system was also verified.' Methods 1* Sampling techniques 1*1 High volume air samples 24-hour samples from about 2000 m3 of air were collected on 8 x 10 MSA 1106-B flashed fired glass fiber filters by means of a General Metal Work HV sampler;: 1*2 low volume air samples ("smoke" samples) 24-hour samples from about 2 m3 of air were collected on 5.5 cm Munktell*s No GO filters, situated in a standard British smoke sampling filter holder of a 1/2" sampling surface by means of a Bymax II A diaphragm pump (IX-3). DUP050311919 IX--2 TUe samplers were situates side by side on a roof of a low building* 2. Preparation of samples for analysis 2.1 High volume samples At first the samples were ashed in a muffle furnace at 500 to 550C according to the reoommonded standard procedure (IX-4). As this procedure proved to cause high losses of lead, it was abandoned and replaced by" the ' California" acid digestion procedure (IX-5). According to this procedure, a mixture of nitrio, sulphuric, and perchloric aoid, and deionized water in the ratio 3.79 t 0.6 * l j 4.79 was used for digestion* Since we did not use membrane filters, the procedure had to be modified in that the solution had to he separated from the glass fiber filter pulp by filtration through a Whatman No 42 filter paper* As this last step may represent a new possibility of lead loss, another upproaoh to the wet "ashing" of samples was also tried and that is the Soviet extraction of samples or aliquots with the redistilled nitrio aoid, and the two procedures were oompared* 2.2 Smoke samples No special preparation of smoke samples for analysis was necessary* Dissolution, separation, and analysis were performed directly on the filter paper used for sampling. DUP050311920 IX-3 3. Analysis of air samples 3.1 Analysis of HV samples 3.1.1 Atomic absorption spectrophotometry The sample extracts (or aliquots) were evaporated to dryness and redissolved in 1# EDTA solution(pH 8) in order to complex lead and thus achieve better atomization.. A.UNICAM SP 90 instrument was used for the analysis. The concentration of lead was read from the calibration curve obtained with standard lead solutions containing 5 to 50 p.g of lead per ml of lc/ EDTA. The optimal conditions for measurement were found to be % lamp ourrent 6 mA, wave length 2170 A. slit 0.2 mm, flame height 0.8 cm, and acetylene flow 1.0 1/min. 3.1.2 The ring-oven method Two 1-cm-diameter disks were cut out of HV samples on glass fiber filters and each disk was fixed face-down with three micro-drops of "UHUB glue (XX-S) and analysed by the chromate method using the ring-oven technique. 3.2 Analysis of smoke samples Smoke samples were analysed by the chromate method using the ring-oven technique. DUP050311921 AGREEMENT BETWEEN TWO INDEPENDENTLY PROCESSED ALIQUOTS ANALYSED.BY ATOMIC ABSORPTION PROCEDURE SAMPLING I S02HLET EXTRACTS O ACID DIGESTS SITE II A y / A/ pgPb/m3 I* A2 A^ 46 JA/V* yr y yw Fig.IS-I I ' j^Fb/a3 DUP050311922 EFFECT OF DIGESTION PROCEDURE ON LEAD RECOVERY AD "ACID DIGESTION, SOX*SGXHLET EXTRACTION EFFECT OF CONCENTRATION ON RESULTS OBTAINED BY ATOMIC ABSORPTION SPECTROPHOTOMETRY DUP050311923 RING-OVEN ANALYSIS AGREEMENT BETWEEN TWO ALIQUOTS OF A HV SAMPLE RING-OVEN ANALYSIS AGREMENT BETWEEN TWO PARALLEL SMOKE SAMPLES DUP050311924 IS--4 Digestion procedures Two pairs of 8 x 10 cm aliquots of the HV samples were processed by both the Soxhlet extraction and the acid digestion and analysed using AAS* There was a very good agreement between the two independently processed aliquots (Fig.IX-l)^ which shows that all the mentioned procedures yield reproducible results* But, by comparing the results obtained by the Soxhlet extraction and the acid digestion it was shown that the latter was yielding systematically lower results (Fig.IX-2). A blank, run with every three samples, did not indicate any contamina tion of the Soxhlet extracts. The reason for the difference in the results obtained by the two digestion procedures may be the loss of lead on filter and glass surfaces in the course of the mixed acid digestion procedure. Analytical methods m order to check the effect of concentration on the atoraiaing efficiency of the flame in the AAS analysis, two aliquots of different volumes (50 and 20 ml) were evapo rated and redissolved in the same volume of 1% EDTA solution (5 ml), thus yielding final solutions of different concentrations. No indication of salt concentration influencing the signal was observed (Fg.IX-3). The two HV sample aliquots analysed by the ring-oven method were in excellent agreement (Fig.lx-4), and the two independently collected parallel smoke samples analysed by the same method gave also very well matched results (Fig.IX--5), showing that the ring-oven method yields reproducible results* xThe straight lines on the diagrams represent a theoretical agreement. DUP050311925 RING-OVEN VERSUS ATOMIC ABSORPTION SPECTROPHOTOMETRY (SOXHLET EXTRACTS) ' ALIQUOTS OP THE SAME HV SAMPLES DUP050311926 CORRELATION BETWEEN THE RESULTS OBTAINED BY RING-OVEN ANALYSIS OP SMOKE SAMPLES AND ATOMIC ABSORPTION SPECTROPHOTOMETRY OP HIGH VOLUME SAMPLES DUP050311927 DAY TO DAY FLUCTUATIONS OF LEAD IN AIR OBTAINED BY DIFFERENT PROCEDURES F ig . IX -9 08 CO to 08 CO 03 DUP050311928 IX--5 The lead concentration obtained by the ring-oven analysis of the HV samples was always lower than the oonoentration obtained by the AAS analysis of Soxhlet extracts (Fig.IX-6), but it was in fair agreement with the concentration obtained by the AAS analysis of acid digests (Fig.IX--7). The lower results obtained by the ring-oven analysis of the HV samples are most probably caused by a poor diffusion through glass fiber filter web. Total procedures % The correlation between the two total procedures, that is between the ring-oven analysis of smoJce samples and the AAS analysis of the Soxhlet extracts of HV samples (Fig.IX-8) was very favourable (r 0,93). If the influence of different hinds of filter media and of a different flow rate on sampling efficiency is t^akea iat account (IX-7), as well as a different way af recording air volume and the faot that each of the two analytical methods measures a different quality of lead, a better correlation may hardly be expected.' Day to day variations of the lead concentration according to the results obtained by different procedures are shown in Fig.IX-9. Reliability of sampling The sampling efficiency, although being a very critical part of air pollution measurement, is very often neglected. In order to put sampling efficiency under control, the sampling systems were regularly calibrated and cheoked for leakage; The air flow was measured at the beginning and at the end of each sampling period, A set DUP050311929 IX-6 of 15 oonseoutive measurements showing a relative doorcase in the flow rate after a 24-hour sampling period is shown in Table IX-1. Table IX-1 HV sample Smoke samples - I II Total Total Relative Total Relative Total Relative weight volume decrease volume decrease volume decrease of of air in air of air in air of air in air sample m3 mg flow *- ... m3 flow *............... ... m3 flow *. ... 147 2043 97,8 3.28 98.8 162 1790 92.8 2.87 93.8 320 1957 92.1 1.76 80*0 292 1920 94,0 2.04 95.9 389 1907 90,0 1,77 . 83.2 298 196 93.0 i.ar Q 283 1904 4,0 1.78 i@,l 040 1950 . 86.0 1.87 87,1 305 1903 95.8 1.79 89.0 329 1870 95.0 1.90 90.0 169 1906 92.0 1.83 91.6 191 1998 96.0 1.90 88.7 245 1964 92.0 1,82 88.1 175 1976 98.0 1.78 88.4 288 1890 88.0 1.71 85.7 2.05 1.82 1.93 2.23 1.88 3.00 1.87 2.03 1.91 2.08 1.93 2.04 1.87 1.88 1.85 98.6 99.4 81.4 98.0 79.2 6.1 83,2 86.2 82.0 88.0 88.0 82.4 76,8 80.0 81.5 The results show that both the low- and the high-volume samplers change the flow rate during the sampling period. Changes in the flow rate were more frequently higher for smoke samples, which means that all the fractions of the day were not equally represented in the.24-hour average of the two samples collected in a different way. The diffe rence was, however, in most oases not too large, although it may have contributed to the scattering of the results obtained by the two procedures. DUP05031 1930 IX-7 Th efficiency of Munktell*s No 00 filters for lead retention was checked by putting a glass fiber filter behind* The experiment was run for 27 days with the same glass fiber filter, while four Hunktell's filters were exchanged* She results are shown in Table IX-2. Table IX-2 VolumegOf air Total amount % of nr of lead jtlg total 1 HunktellNo 00 !. 2. 3. 4. MunktellJs 1 - 4. II Glass fiber filter Munktell's + glass fiber 11.3 10.9 10.6 10.8 43.6 43.. 6 43.6 ..... 4.0 4.0 3.5 5.0 16.5 1.8 18.3 21.9 21.9 19.1 27.3 90.1 9.9 100.0 The retention effioieaoy of Mhaktall'a No 00 filter is about 90%. The results reported earlier were not corrected in accordance with this finding. In further investigations a number of parallel EV and smoke samples are planned to be oolleoted at various sites differing in the quantity and composition of air-borne particulate matter, and analysed by both the AAS and the ring-oven method, for the final evaluation of the latter. Should the ring-oven method prove to be reliable under all conditions, this would allow the determination of lead in air within a large scale survey, since the method is fast and simple, fairly accurate and reproducible, and requireing inexpensive equipment for sampling and analysis. DUP050311931 References 12--X Weisz,H,, Mikrochim. Acta, 1954. 140. 12-2 Fugas,M. and Paulcovid,R., Anal.Claim.Acta, in press. IX-3 Methods of Measuring Air Pollution, Report of the Working Party on Methods for Measuring Air Pollution and Survey Techniques, OECD, Paris 1964. 12-4 12-5 Jutze,G.A.' and Poster,K.E. , J.Air.Poll.Control Assn. 1' 7 (1967) 17.: * Zimmer, C.fE.' and Morgan, G.B., APCA Paper 67-152, Annual Meeting of the Air Pollution Control Assotia- tion, Cleveland 1967. 12--6 West,P.W., Weisz,H., Gaeke,GvC. and Xyles.Gv-, Anal.* Chem., 32 (1960) 943. 12-7 LeClare,P.C.1, Breslin,A.J. and Ong,L.D.X., Am.Ind. Hyg.Assn.'J., 30 (1969) 386. DUP050311932 X DETERMINATION OF LEAD IN BLOOD In 1952 a "mono-colour" dithizone method for determi nation of lead in Blood was developed in our laboratory (X-l). The special characteristic of the method was the elimination of iron with cupferron prior to dithizone extraction. The method was used for the determination of normal values of lead in blood in the population of Zagreb and some rural districts, and for the determination of lead in blood of occupationally exposed subjects, and showed to be very reliable. For each determination two parallel analyses, requi ring 5 ml of blood each, have to be run, which means that only for lead determination 10 ml of blood would be necessary from each subject. In the frame of this investi gation an attempt was made to modify the method in such a way that smaller volumes of blood oould be used for the analysis* In the modified procedure one or two ml portions of venous blood are put into a KJeldahl flash and digested with several 3 to 5 ml portions of double distilled nitrio acid allowing each portion to evaporate to dryness before the next one is added. After the content of the flash becomes clear, the digestion is continued with two or three portions of hydrogen peroxide. The dry residue is dissolved in double distilled water, transferred into a separating funnel, the iron is precipitated with cupferron (2% water solution) and the iron oupferrate is extracted with carbon tetrachloride. Aqueous layer is again evaporated to dryness with addition of nitric acid in order to destroy the excess of cupferron. The residue is boiled with 10 ml hydroxylamine hydrochloride solution (20 g in 100 ml), 10 ml of DUP050311933 X--2 citrate solution are added (20 g per 100 ml), a few drops of ammonia to 'bring the pH to 10.5, and then 15 ml of a pH 10,5 Puffer (2 g of citric acid, 4 g of potassium cyanide, 2 ml of double distilled ammonia and double distilled water up to 100 ml). Lead is extracted with one 5 ml portion followed by a few two ml portions of dithizone solution (0.001$ in carbon tetrohloride). The organic phase is collected in another separating funnel and shaken with 40 ml of potassium acetate buffer (pH 4.5 - 5) in order to destroy lead dithizonate and bring lead back into the aqueous phase. The so isolated lead is again extracted with the dithizone solution after the pH of the aqueous phase has been set to 10.5. Ttie lead dithizonate extracts are combined, separated from water by centrifugation, made up to 10 ml with earbon tetrachloride and the absorbance of the solution is measured against blank at 520 mm in 1 cm cells with a Beckman Model DU spectrophotometer. The procedure has been applied to one and two ml aliquots of a blood sample with addition of various amounts of a lead standard. The results obtained with one ml aliquots were very scattered, but those obtained with two ml aliquots were in favourable agreement with the expected values. In the first part of our field investigations the new modifi cation of the procedure will be verified against the original method and if reliable results are obtained, it will' be used in the further investigation. References X--1 Weber,0.A., Voloder}K. and 3 (1952) 296. DUP050311934