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IStrfl tel / Reprinted from the A. M..A. Archives of Industrial Health October 1955, Vol 12, pp. 396-405 Copyright 1955, by American Medical Association in csCead ~sdldorpti ion J. WYUIE, M.A., M.D., B.Sc., D.P.H. Kingston, Ont., Canada s'** * 1 1. INTRODUCTION There is a great need for a simple chem ical test which will give- an early indication of dangerous lead absorption or incipient lead' poisoning. Quantitative analyses of urine for lead require specific and expensive equipment besides trained personnel, and the methods are tedious and time-consuming. Enumeration of stippled cells in blood films is- laborious and hpt entirely specific. The newer knowledge of porphyrin metabolism resulting from the work of Watson in America, Rimington in Britain, and Wal denstrom in Sweden has shown the need for a careful evaluation of urinary prophyrins in health and disease. 2. DEFINITION AND DESCRIPTION . OF PORPHYRINS s lifetrphyrins are pigment compounds which are widely distributed in nature and consist chemically of four pyrrole rings linked together by four methene (CH) bridges. The pyrrole ring has the configuration shown in Figure 1, and four such rings joined together by methene bridges form porphin (Sig. 2). In the naturally occurring porphy rins, the eight hydrogen atoms of the pyrrole rings are substituted by methyl (CHa), ethyl (QHb), vinyl (CH2.CH), propionic acid (CH2 CH2COOH), and acetic acid Submitted for publication Jam 31, 1955. Supported by a National Public Health Research Grant - Professor of Preventive Medicine, Queen's University. Presented at: the combined meeting of the Sec tion on Industrial Medicine of the Ontario Medical Association and the Industrial Medical Association of the Province of Quebec, Ottawa Civic Hospital, Sept. 25, 1954. HC ;h Ni/ H PYRROLE Figure 1 (CH2COOH) groups. Coproporphyrin may exist in four isomeric forms, depending on the sequence of methyl (M) and propionic acid (P) groups, and since only Types I and III isomers have so far been isolated in clinical conditions, it will suffice to represent them in Figure 3. The uroporphyrin isomers contain acetic acid (A) and propionic acid (P) groups, and Types I and III isomers are represented in Figure 4. The terms coproand uro- porphyrin were introduced by Fischer 6 and imply that these pigment com pounds occur separately in the feces and urine. It is now known that they occur normally as well as in pathological conditions in both excreta. 3. CHEMICAL PROCEDURES (a) Qualitative Test for Urinary Copro porphyrin,--According to de Langen and ten Berg 1 the detection of coproporphyrin in abnormal amounts in the urine is a more reliable method than the degree of basophilic stippling of erythrocytes in the early diag nosis of lead poisoning. The method is simple and rapid and can be made semiquantitative as a screening test where large numbers of workers are exposed to lead dust and fume. A small sample of urine (S, 10, or 20 ml.) is acidified with a few drops of glacial acetic acid, a few milliliters of ether (2 to 5 ml.) |||||K H iiJijji lii* Bllii till N36906 2 added,'and, after shaking the stoppered tube, the ether layer is inspected under an ultra violet', lamp. A blue, pink, or red fluores cence may be observed. In'' searching the literature, no less than seven modifications of the original de Langen and ten Berg test were noted. The modifica tions are summarized in Table 1 and show the quantity of-urine taken for the test, the reagents added, the procedure, and the scheme for treading the result according to the intensity of the fluorescence under an ultraviolet lamp. Meek, Mooney, and Harrold suggested the addition of 3% hydrogen peroxide (2 drops) to change any interfering substances which might cause doubtful reactions. In my experience, ether alone gives a blueviolet fluorescence under ultraviolet light, and the use of hydrogen peroxide is seldom necessary. Other solvents besides ether, such as isopropyl ether, ethyl acetate, and amyl =CH s PORPHINIE) Figure .2 65 SHORT-HANO FORMULA FOR PORPHiN Ta b l e 1.--Madifixations of Test for Coproporphyrin Method Qaantity o (Brine (or Test, 1. de Langen and ten Berg1 2. Meek, Mooney, and Harcold 8 20 8. WaWman and 4. Maloof 8 5. McCord 10 6, Brooks10 7. London School of Hygiene and TpopEcal Medi cine * Reagents Added Few drops glacial acetic acid; % ml. ether 2 ml. glacial acetic acid; 2 drops 8% HsOs; 2 ml. ether 2 drops glacial acetic acid; 2 drops 8% HsOa; 1.5 noL ether 6 drops 6 N acetic acid; 5 ml. ether 2 drops glacial acetic add; 2 drops $% HsOs; 2 ml. ether 1 ml. glacial acetic add; 5 ml. ether; 3 drops HaOs 2 ml, glacial acetic add; 20 ml. ether 8* U. 8. Naval Shift- 5 yard Method xx 8 drops %% HsOs; 6 drops glacial acetic acid; 5 ml. ethylether Procedure Shake several times; examine ether layer under ET.VJE*. Shake several times; examine ether layer under U.V.L. Shake stoppered tube on long axis 20 times; examine under 'Wood Lamp Shake and vtew in dark room with Black Light Lamp Stopper#* ml. test tube; shake on long axis 20 times; allow layers to separate and read after 10-15 min. Invert tube several rimes; allow to stand for 10 min. Shako 1 sum.; discard lower layer; wash ether twice with D.W.; shake with 0.25% HC1 In lots of 2, 2, and 1 ml.; adjust arid extracts to G ml. in Mohaae tube Stopper tube and shake SO see.; allow layers to separate; ex amine under U.Y.L. Sherwood, B. J.; Personal communication to the author. Positive Result Bose to deep red Light pink to red fluorescence Fluorescence in ether layer Fluorescence with various shades of red Bed fluorescence; slight, moderate, marked, and. very marked Violet -f Fink ss -HLigbt rose = , . , Deep rose = -H'4-h Match against tube containing standard soL in fluorescence comparator Fink to orange-red fluorescence * .1 i- -fl', w"' S*-W S'V*S*.*VrI, DUP050312371 i&i^iliiikMuiii'ii'ii the violet changes to faint blue and at pH 9 and pH 10 to a blue coloration indistinguish able from that obtained with normal.urine. Since urine contains uroporphyrin as well as coproporphyrin, the blue coloration is due to its presence when the coproporphyrin has been suppressed in the alkaline range. (b) Quantitative Determination of Uri nary Coproporphyrin and Uroporphyrin.-- The determination of coproporphyrin quan titatively requires the following: acetate, have been tried, but the best solvent is ethyl acetate which of itself does not show any fluorescence under ultraviolet light. I use the following test: To 2.5 ml. of urine in a test tube, 3 to 5 drops of 6 N acetic acid are added to bring the urine to pH 6, testing with hydrion test paper of range 1 to 11. Two milliliters of ethyl acetate * is added; the stoppered test tube is inclined on its tong axis 10 times (violent shaking is unnecessary) and allowed to stand for two or three minutes. The fluorescence of the ethyl acetate layer is observed under radiation from an ultraviolet lamp.f The addition of acetic acid, not hydrochloric acid, is necessary to permit the ethyl acetate layer to extract coproporphyrin from the urine. If hydro chloric apid is used, the ethyl acetate layer appears a greenish blue at pH 6 under ultraviolet light, and this coloration persists at pH S, pH 4, and pH 3, while at pH 7 up to pH 12 the coloration is blue. With use of the technique described, it has been found repeatedly that a violet or heliotrope fluorescence in the ethyl acetate layer under ultraviolet light corresponds to approximately 5y of coproporphyrin per 100 ml. of urine. It ean be shown that the intensity of the fluorescence varies with pH, for at pH 5 the violet changes to pink and this, in turn, changes to red at pH 4 and pH 3. At pH 8, (i) Use of a fluorescence attachment to the Uvispek Spectrophotometer, with a sensi tivity ujwn to 0.57 per. 100 ml. of 1.5 N HO (ii) Preparation of standard solutions of cop roporphyrin $ (Hi) Preparation of calibration curves (Fig. 5), plotting coproporphyrin concentration (micrograms per 100 ml.) against per cent transmission (% T) In Schwartz, Zieve, and Watson's method, 5 ml. of urine is pipetted into a Squibb sepa ratory funnel, 5 ml. of buffered acetic acid added, and the coproporphyrin removed by extracting with ethyl acetate. The method permits the removal of ether-insoluble por phyrins by washing with 1% sodium acetate and the conversion of precursors to porphy rins by shaking with dilute iodine solution. The coproporphyrin is finally extracted from the ethyl acetate in 1.5 N HC1 (20 ml.). The per cent transmission is measured in the spectrophotometer, and the coproporphyrin concentration estimated from the graph of t Supplied by Dr. S. Schwartz of the University of Minnesota. y * Ethyl acetate CHa COO CaHs has the same price as ether (ethyl ether) [(GHs)aO]. t The Hariovia mercury-arc Inspectolite (Hanovia Chemical & Manufacturing Co., Newark, N. J.) is eminently satisfactory. This lamp has a special dark-glass filter, practically opaque to vis ible fight generated in the mercury-arc quartz tube, hut freely transmits light in the region of 3660 A., tte' ftiost powerful wave lengths causing fluores cence AA PP PA r UROPORPHYRINS Figure 4 PA EL lifiliiliiiii l|P Ml ran IlflilP !i|||| ifiill Bjf Mil I mm ill Slllil Sh lilSi DUP050312372  : , 4 10y per 100 ml. or of lOOy per 100 ml. of 1.5 N HC1 solution.' Uroporphyrin has been determined quan titatively by Rimington's method -which consists in calcium chloride precipitation of uroporphyrin from a small amount of urine, in the presence of alkali and excess calcium ions, and its subsequent measurement in the Soret band region. The methods used for these estimations have been as follows: An apparatus has been constructed, with the aid of the University instrument maker, in which an AH 4 mercury-vapor laihp (supplied by the Gen eral Electric Co., Toronto), as a source of suitable wave lengths of ultraviolet light, has been inserted in a metal housing (manufactured by Hilger & Watts, London), with a condensing lens to trans mit "parallel" light. The primary filter consists of a 405 mv- interference filter (obtained from Baird Associates, Cambridge, Mass.) combined with a References; 4 atid 5. blue auxiliary filter (Corning Auxiliary Filter 4-72). The combination of the interference filter, transmitting 30.5% of the 405 mp. exciting light, with the blue filter,- which has a transmission value of 52% at 406 m/, permits 19% of the light to be transmitted. A secondary red filter (Corning 2-61), which transmits light above 620 ttu* and has negli gible transmission below 600 aw, is inserted in front of the photoelectric cell of the Uvisnek Spectro photometer. With this combination of filters, a blank solution of 1.5- N HC1 gives only a minimal deflection of 0.5 mm. When a 10V solution of copro porphyrin I is set to read 100% T. This gives the sensitivity of the Uvispek. The intensity of fluorescence is determined by inserting a quarts cell containing 1.5 N HC1 as a blank into the adapter of the Uvispek, setting the transmission at zero, and centering the galvonoroeter needle by means of the zero knob on the potentiom eter of the Uvispek. The porphyrin standard is next set to give a transmission reading of 100 by adjusting tbe check knob, The transmission reading of the unknown is then determined. (No use is made of the light source or monochromator of the UVispek Spectrophotometer in fluorescence deter minations.) Fig. 5.--Calibration curves. A, curve prepared by plot ting average per cent trans mission readings against coproporphyrin concentrations 5t to lOOy per 100 ml. of 1.5 N HO. B, curve pre pared by plotting average per cent transmission read ings against coproporphyrin concentrations 0.57 to lOy per 100 ml. of 1.5 N HC1. m jg*4gp i%a V T.I H DU P050312373 SIPIS mam 4. EEStji.^S '!&F`POH^BrifitlN: DETERMINATIONS. ; ON URINS SAMPLES The urine samples were analyzed quanti tatively from the following; (i) Presumably normal, healthy individuals (), Employees with a: mild exposure to lead , , ,' in an industrial process, e. g., repair of motor-vehicle bodies and glazing o wall tiles '; (Iff)' fiinplbyees liable to a severe exposure to ! 'lead in a storage-battery plant (i) To obtain data on the values of urinary coproporphyrin and uroporphyrin excreted normally, ' morning urine samples were obtained from fourth and fifth year medical students, during March and April, 1954. Sjbtty^six samples were examined. Qualita tive tests for coproporphyria, for urobilinogen and ' porphobilinogen using Ehrlich's alde hyde reagent, for urobilin using Schlesinger's test, and for bile pigments using Ehrlich's diazb reaction were performed prior Normal urine samples 12 Coproporphyria Uroporphyrin flange:0'834-80 #<g% Range; 125--9-57 pq% Figure 7 to the quantitative determinations of porphy rins. On tabulating the results, it was,noted that urines with strongly positive reactions for urobilinogen had higher uroporphyrin values than urines with negative or weakly positive reactions for urobilinogen. Twentytwo out of sixty-six samples had this feature and were tabulated separately. The remain ing samples were selected at random, and two groups of 22 samples formed. Figures 6 and 7 show the range of values for the first and second normal groups and Figure 8 the range for the group with high uroporphyrin values. The results are given in Table 2. The average uroporphyrin value for the third group is approximately three times that for either the first or the second group, while the average coproporphyrin value is only slightly increased over that for the first or the second group. (it) Six healthy men; aged 54, 23, 34, 40, 38, and 23 years, respectively, were DUP050312374 Ta b l e 2.--Resttlts of Porphyrin- Determinations on Urine Samples Group m- Normal 2d ' Normal m Normal with high uropor phyrin Values Range Average Range Average Range Average Copro porphyria, 7/100 Ml. 0.65 --15.16 2.76 dr H>.8S 0.83 -- 4.80 2.80*0.28 1.06 -- 7.13 3.31 0.83 Uro porphyrin, 7/100 Ml. 0.92 --10.07 5.45 0.61 1.25-- 9.57 5.52* 0.53 11.44 -- 25.18 15.97* 080 allowed to begin work in an industrial proc ess involving exposure to lead dust. Ted days later the collection of weekly samples Normof urines with high Uroporphyrin of urine was commenced and continued for six weeks, the period extending from Sept. 9 to Oct. 16, 1953. The urine samples were collected in chemically clean bottles of 16 oz. capacity, each containing 0.5 gm. of pure, dry sodium carbonate. The'men were instructed to pass urine directly into the bottles on rising at 7 a. m. on Wednesday of each week. Simultaneously, blood films for the estima tion of stippled cell counts || were taken weekly from each workman. So far as known, the workmen had not been engaged previously in an atmosphere where there was a lead hazard, and blood films taken prior to employment showed negligible stippled cell counts. The new type of work consisted in repair ing motor-vehicle bocKes in which buffing and spray painting were executed. Due pre cautions in wearing respirators and pro tective clothing were observed, so that the exposure to lead was considered minimal. Coproporphyrin determinations were car ried out on the urine samples on the same |[ The blood films were stained and the stippled counts estimated in the Industrial Hygiene'Labora tory of the Ontario Department of Health. Ta b l e 3.--Porphyrin Determinations on Morning Urine Samples Submitted Weekly from Three Workmen with Light Exposure to Lead Copreporphyrin Uroporphyrin Figure 8 Date Copro- 4/14/54 286 4/20/54 2.82 4/28/54 3.91 5/ 6/54 4.49 5/12/64 3.06 5/19/64 1.71 5/28/54 6.76 6/ 2/54 6/10/54 7.18 .... 6/18/54 7.20 6/23/54 3.74 11 1/U 3.82 7/14/64 6.30 7/22/54 7/28/54 6.78 8/13/54 3.10 B/19/64 4.12 8/27/64 3.03 Average 4.4 values Uro- 6.67 6.72 1.14 1.87 2.29 0.46 2.66 7.99 j.. 5.04 2.52 2.98 5.20 .... 6.04 4.10 6.27 1.87 3.3 1 Oopro- 3.16 4.51 ..... 1120 6.86 4.07 11.32 11.7 8.62 6.02 6.90 6;94 6.44 7.B6 4.5S 4.06 7.32 7.15 too-* ' Copro- 6.18 2.13 6.49 8.88 .... 6.41 8.60 8.06 9.15 8.56 0,21 4.50 3.43 7.78 5.04 8.10 12.10 4.14 4.74 5.00 4.85 6.34 12.80 6.03 6.87 7.01 8.70 6.87 6.46 3.78 4.22 2.69 4.28 16.60 6.55 Tjro-' 8.24 6.41 5.04 2.62 8.70 1.66 2.60 9.15 4.12 3.49 2.06 6.49 4.81 1.46 2.52 8.63 4.8 or DUP050312375 7 .{Jsy/fOr qn the following day after being received in the laboratory. The 36 observa tions on coproporphyrin content and stippled cell counts were plotted as shown in Fig ure 9. Twenty-two observations, with counts ' less , than 1000 stippled cells per 1,000,000 , erythrocytes, had a range front 0.6y to 7.5y of weekly samples of urine examined for their porphyrin content. In all, 16 weekly samples were examined from April to August, 1954, and the average values calculated, as shown in Table 3. During this period the stippled cell counts were less than 1000 per 1,000,000 erythrocytes. Vi' fVr --1---------------- _---------,----------------- -5-----------------,---------------------------------------j------------------- ------------- ,-- Urine somples from R. Ci C. M, E 1, - , , ... V( fj, o - mmmm t> * * ' if Eft, 8 -& 0 r1 9 O o O - 00 3 -B 8 i <1000 (22) 8 O o 3O 1 1000 (10) 11 1500 <31 Stippled* Cells per 10* r b cells Figure 9 1 2000 - - 1 2400 (t> coproporphyrin per 100 rbl. of urine, the average value being 2.69y0.35y per 100 ini. Thirteen, observations, with counts of 1000 to tSdO stippled cells per 1,000,000 erythro cytes, had a range from 0.5y to 11.6y of coproporphyrin per 100 ml. of urine, with an average value of 3.54:0.86. Three ct these workmen were followed for a subsequent period of four months, and ( It may be noted that, although there are wide variations in the weekly determinations, the average coproporphyrin value's for J. R. G., W. V., and E. E. are higher than the average value for counts between 1000 and 1500 stippled cells per 1,000,000 eryth rocytes (4.4, 7.15, "and 5.55 as compared with 3.54y per 100 ml.). There is, neverthe less, an indication that the coproporphyrin Bi S8K- DUP050312376 8 values increase as the stippled cell counts increase from less than 1000 to 1000 arid 1500 stippled cells per 1,000,000 erythro cytes (Fig. 9). The uroporphyrin determinations have also been carried out weekly, and the average values calculated. The copro-/uro- porphy rin ratio is on the average 1:1, although there are wide differences in the individual ratios. <1000 Roftga:! 2 -2& 7 Coproparphyrin 4COO ieiffo-i * Range. I 3 '46 6 Uroporphyrin Figure 10 A number of workers with a mild expo sure to lead in a tile-manufacturing plant where the tiles are sprained with a lead glaze before firing in kilns, have been under medi cal supervision for a number of years'. Morn ing urine samples obtained weekly on several occasions have been analyzed for porphyrin content. As blood films for stippled cell counts are taken only at three-month inter vals in this plant, the porphyrin determina tions on urine samples, submitted the next day after the blood films were taken on Aug. 18, 1953, and on March 16,3954, were considered suitable for study of a correlation between stippled cell count and amount of urinary coproporphyrin. Twenty-eight urine samples had a coproporphyrin range from 1.2y to 28.7y per 100 ml., the average value being 10.862*1.316, and a uroporphyrin range from 1.3-y to 46.6y per 100 ml., with an average value of 10.57*2.012. Figure 10 shows the results graphically. All these employees were reported to have counts less than 1000 stippled cells per 1,000,000 erythrocytes. It may be noted that these values are dis tinctly higher than those obtained in the study of employees exposed to lead dust in the repair of motor-vehicle bodies. The com parison, however, is not valid, since the tile workers had been exposed for several years in the tile plant. Their average values may be considered therefore to provide a norm for persons working for several years in an atmosphere with a mild exposure to lead. (m) A number of urine samples were obtained from employees in a storage-battery manufacturing plant on the morning follow ing the day when blood films were taken for stippled cell counts in August, 1953, and April, 1954. Six employees had stippled ceil counts less than 1000, two had counts of 1000, and seven had counts varying from 1600 to 9800 per 1,000,000 erythrocytes. Figure 11 shows that there is a definite tend ency for increased coproporphyrin excretion when stippled cell counts exceed 1000 per 1,000,000 erythrocytes. The range for coproporphyrin excretion with stippled cell counts of 1000 and less than 1000 per 1,000,000 erythrocytes was 3y--120y per, 100 ml., the average value being 55.21*15.04. For uroporphyrin, the corresponding range was 2.5y--75.15y per 100 ml., the average value being 34.52*9.29. These average values are dis tinctly higher than those obtained in a mild lead industrial exposure. It is interesting to note the copro-/uroporphyrin ratio, which has not been referred DU P050312377 PW.1 to previously in the literature. With stip (ii) An attempt has been made to cor pled cell counts of less than 1000 and counts relate stippled cell counts with quantitative of 1000 per 1,000,000 erythrocytes, the coproporphyrin determinations in a group of copro-/uro- porphyrin ratio seldom exceeds employees mildly exposed to lead for the 2:1, but as the stippled cell counts increase, first time in buffing and spray painting of the copro-/uro- porphyrin ratio becomes damaged motor-vehicle bodies. As the stip 3:1, 4:1,.5:1, and 6:1, as indicated in pled cell counts increase to 1000 and 1500 Table 4. per 1,000,000 erythrocytes, the coproporphy rin values tend to increase. With continuing Tabl e 4.--Change in Ratio of Coproporphyrin and mild exposure to lead, although the stippled Uroporphyrin, with Increase in Stippled Cells cell counts may not exceed 1000 per 1,000,000 Name T. H. P. B. Tob. Top. A. O. Stippled Cells 4,000 1,000 1,600 1,600 5,800 3,700 5,300 0,800 Oopro- Uro porphyrin, porphyrin, Y/lOO Ml. *y/100 Ml. 60.0 27.9 B8.0 44.52 231.0 37.0 181.0 144.0 509.0 1,831.0 70.5 12.0 46.3 28.0 66.3 216.0 O/U 2:1 2:1 s:l 3:1 4:1 b :i 5:1 6:1 erythrocytes, the urinary coproporphyrin val ues tend to increase. Employees with a mild exposure to lead for several years have higher urinary copro porphyrin and uroporphyrin values than those found in normal persons. (in) Employees liable to a severe expo sure to lead show high values for urinary coproporphyrin as the stippled cell counts exceed 1000 or 1500 per 1,000,000 erythro 5. SUMMARY AND CONCLUSIONS cytes. The uroporphyrin values are also (i) In porphyrin analysis of morning increased. It is interesting to note that in urine samples from apparently normal per this type of exposure the copro-/uro- sons, it has been observed that urines with porphyrin ratio increases from the normal a strongly positive qualitative test for uro 1:1 or 2:1 to 3:1 and may reach 6: 1. bilinogen generally have high uroporphyrin values. There is evidence that this feature occurs in persons on a high-vegetable diet as well as in certain liver diseases. REFERENCES 1. de Langen, C. D., and ten Berg, J. A. G.: Porphyrin in the Urine as First Symptom of Lead Poisoning, Acta med. scandinav. *80:37, 1948. DUP050312378 10 2. Meek, S. F.; Mooney, T., and Harrold, G. C.: Urinary Porphyrins in Lead Poisoning, Indust Med. 17:469, 1948. 3. Schwartz, S.; Zieve, L., and Watson, C. J.: Improved Method for the Determination of Urinary Coproporphyrin and Evaluation of Factors Influ encing Analysis, J. Lab. & Clin. Med. 37:843, 1951. 4. Svemsson, S. L.; Rimington, C., and Barnes, H. D.: Complete Porphyrin Analysis of Pathologi cal Urines, Scandinav. J. Clin. & Lab. Invest. 1:2, 1949. 5. Rimington, C., and Sveinsson, S. L.: Spectrophotometric Determination of Uroporphyrin, Scahdinav. J. 'Clin. & Lab. Invest 2:209, 19S0. & Fischer, H., and Orth, H.: Die Chemie des Pyrrols, Kothen, Germany, Paul Dunnhaupt, 1937, Vol. 2, Pt. 1. 7. Waldman, R. K., and Seideman, R. M.: Reliability of Urinary Porphyrin test for Lead Absorption, Arch, Indust Hyg, & Occup. Med. 1:290, 1950. 8. Maloof, C C.: Role of Porphyrins in Occu pational Diseases: Significance of Cbpropqrphyrinuria in Lead Workers, Arch. Indust. Hyg. & Occup. Med. 1:296, I960. 9. McCord, C. P.: Porphyrins: Significance in Occupational Diseases, Indust Med. 20:185, 1951. 10. Brooks, A. L.: Appraisal of a Urinary Por phyrin Test in Detection of Lead Absorption, Indust Med. 20:390, 1951. 11. Correlation Between Urinary Lead Concen tration and Urinary Porphyrin Determinations, M. News Letter (U. S. Navy) 22:35-36, 1953. 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