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CLIN. CHEM. 21/1. 93-97 (1975) 

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Zinc Protoporphyrin (ZPP): A Simple, Sensitive, 

Fluorometric Screening Test for Lead Poisioning 

Angalo A. Lamola,1 Morris Joselow,2 and T. Yamane1 

The predominant fluorescent porphyrin that appears in 

erythrocytes as a result of chronic lead absorption or 

iron deficiency anemia is zinc protoporphyrin. This compound can be fluorometrically assayed in diluted whole 

blood wivh high sensitivity. An extremely simple and 

rapid test for lead absorption is described that is based 

on this assay. Results of a field trial indicate that this 

test should be seriously considered as a primary 

screening test for monitoring the effects of chronic lead 

absorption. 

Additional Keyphrases: toxicology • trace elements 

• iron-deficiency anemia • porphyrias 

The massive campaigns now underway in this 

country to detect lead poisoning in children have 

created an urgent need for a practical screening test, 

one that is simple, reproducible, cheap, and, of 

course, diagnostically valid. Direct analyses of blood 

for lead is seriously handicapped by the requirements 

of expensive equipment, skill in analytical technique, 

and, above all, by the scrupulous care demanded to 

avoid the risk of sample contamination from the 

ubiquitous lead in the environment. 

In recent years, such efforts have instead been directed toward measurement of some of the metabolites (or their associated enzymes) resulting from the 

interference by lead with heme synthesis, such as 

{-aminolevulinic acid, t$-aminolaevulinate dehydratase (EC 4.2.1.24), or coproporphyrin (1, 2). The 

most likely candidate to have emerged as a useful 

"metabolic sentinel of lead poisoning" is extractable 

protoporphyrin in blood (3-7). 

1

 Bell Laboratories, Murray Hill, N. J. 07974. 

2

 Departments of Preventive Medicine and Pathology, College of 

Medicine and Dentistry of New Jersey, Newark, N. J. 07103. 

Received Sept. 18,1974; accepted Oct. 15,1974. 

The increase in "free erythrocyte protoporphyrin" 

associated with lead intoxication has been recognized 

for more than 40 years (8, 9). It endows erythrocytes 

with a red fluorescence when excited with blue (400 

nm) light (10), and it is extractable from the erythrocytes as protoporphyrin with acidic solvents (11) 

(such as a mixture of ethyl acetate, acetic acid, and 

HCI). The extracted protoporphyrin can be assayed 

by absorption spectrophotometry or, with greater 

sensitivity, by fluorometry. Both the presence of fluorescence in erythrocytes, observed microscopically 

(12), and the presence of acid-extractable protoporphyrin (3-7, 13-15) have been used as monitors of 

the effects of lead intoxication, but neither test is 

highly specific: an increase in the protoporphyrin 

and, concomitantly, in the red fluorescence, is also 

associated with several other disorders—erythropoietic protoporphyria, iron deficiency anemia, and hemolytic anemias (13, 16, 17). Furthermore, the relationship between the concentrations of the protoporphyrin and of lead in blood and the factors governing 

that relationship are as yet unclear. 

Apparently overlooked in the flurry of reported 

studies (3, 6) on the use of erythrocyte protoporphyrins are some of the basic chemical and spectrofluorometric aspects of the porphyrins. Recent results 

concerning the nature of the fluorescence exhibited 

by the iron-free protoporphyrins of erythrocytes3,4 

—easily detectable in the hemoglobin fraction or the 

whole blood—leads us to propose the use of a much 

simpler procedure based on zinc protoporphyrin3 

(18) for detecting persons affected by lead intoxica3

 Lamola, A. A., Yamane, T., Piomelli, S., Poh-Fitzpatrick. M., 

and Harber, L., unpublished results. 

4

 Piomelli, S., Lamola, A. A., Poh-Fitzpatrick, M., Carlos, E., 

Seaman, C, and Harber, L., unpublished results. 

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WAVELENGTH (nm) 

Fig. 1. Fluorescence spectra of 400-fold diluted whole blood 

The dashed curve is the spectrum obtained from blood ot patients with lead 

intoxication (Pbl). The solid curve is the spectrum obtained from patients with 

erythropoietic protoporphyria (EPP). The two main bands in each spectrum 

are the 0' - * 0 and 0' - » 1 vibrational transitions associated with a single 

porphyrin emission and do not indicate that two types of porphyrin are fluc-

. rescing in each case. The emission from Pbl blood is that of zinc protoporphyrin. The main fluorescence from EPP blood is characteristic of metal-free 

protoporphyrin. EPP blood usually shows some zinc protoporphyrin fluorescence (the shoulder near 590 nm) 

tion. This compound, the fluorescent form of protoporphyrin associated with lead poisoning (18), can be 

measured directly and simply in diluted whole blood, 

and its concentration can be directly related to high 

lead concentrations in the blood of both children and 

adults. 

Methods and Materials 

Analytical Principles 

When blood samples from patients with erythropoietic protoporphyria, lead intoxication, or iron deficiency anemia, are acid extracted in the usual manner (6) the final hydrochloric acid solutions exhibit 

identical fluorescence spectra with a maximum at 606 

nm5 (the excitation maximum is near 405 nm). This 

well-known spectrum is characteristic of protoporphyrin IX in acidic aqueous solutions. In contrast, 

the fluorescence spectrum of whole blood or the hemoglobin fraction from patients with erythropoietic 

protoporphyria is quite different from that obtained 

from the blood or hemoglobin of the latter two categories of patients3

 (18). The spectra are shown in 

Figure 1. Their blood [diluted 400-fold with saline (9 

gl/liter)-phosphate buffer (10 mmol/liter, pH 7.4)] or 

hemoglobin exhibits a fluorescence maximum at 625 

nm (excitation maximum, 397 nm). This fluorescence 

is attributable to protoporphyrin IX in its free-base 

form, bound to hemoglobin at an unknown site(s).3 

In contrast, the whole blood or hemoglobin from 

lead-intoxicated or iron-deficiency anemic patients 

fluorescence maximally at 594 nm (excitation maximum, 424 nm). This fluorescence is due to zinc protoporphyrin IX (ZPP), which probably occupies sites of" 

heme in a small fraction of the hemoglobin; i.e., the 

fluorescent porphyrin in blood from these two kinds 

of patients is not "free porphyrin"3 (18). Bloods containing uroporphyrins or coproporphyrins, or both, 

such as those obtained from patients with porphyria 

cutanea tarda exhibit fluorescence maxima at wavelengths greater than 615 nm.6

 Thus, by avoiding acid 

extraction of the blood, the spectrofluorometric measurement of ZPP allows one to differentiate easily between lead intoxication and iron-deficiency anemia 

and the porphyrias, even erythropoietic protoporphyria.3,6

 Current procedures for measurement of the 

free erythrocyte protoporphyrin do not permit this 

differentiation. 

Spectrofluorometry of ethanol or acetone extracts 

of whole blood also allows differentiation between 

lead intoxication and the porphyrias3 (18). Ethanol 

or acetone extracts the protoporphyrin from the 

blood of leadrintoxicated persons together with its 

chelated zinc. ZPP in ethanol or acetone fluoresces at 

589, whereas protoporphyrin in ethanol or acetone 

(e.g., extracted from protoporphyric blood) fluoresces 

at 632 nm. However, the efficiency with which ethanol extracts these porphyrins from whole blood 

varies—it is 50-80%. Extraction with acetone is even 

less efficient. To increase the efficiency of extraction 

with acetone, Chisolm et al. (3,19) used acidified acetone. When this is used to extract lead-containing 

blood, a mixture of ZPP and free protoporphyrin is 

obtained, because this acid solvent can remove the 

zinc from ZPP. Thus the use of organic solvents in 

such analyses for blood porphyrin is evidently more 

complicated and less quantitative than simply performing spectrofluorometric measurements on diluted whole blood. 

Apparatus 

Fluorescence was measured with a Hitachi PerkinElmer MPF2 spectrofluorometer equipped with a 

Hamamatsu 446 UR tube. With this instrument, the 

fluorescence of unextracted porphyrins in whole 

blood could be measured down to normal concentrations (50-80 fig/liter). Wavelength maxima can be 

measured to ±1 nm. Other commercially available instruments such as the Aminco-Bowman and Farrand 

MK-1 spectrofluorometers, equipped with a similar 

photomultipHer detector, are sufficiently sensitive 

and resolve well enough to give comparable data. 

Lead in whole blood was determined with a Perkin-Elmer Model 403 atomic absorption spectrophotometer equipped with a deuterium background corrector. The 283.3-nm resonance wavelength was used 

throughout. 

5

 This refers to the maximum of the lowest wavelength band in 

the spectrum, as measured with a red-sensitive photomultipHer 

tube as the detector. 

6

 Lamola, A. A., and Poh-Fitzpatrick, M., unpublished results. 

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Procedure 

Because the spectrofluorometer that we used detects fluorescent light at an angle of 90° to the direction of the exciting light, the blood was diluted 500-

fold to avoid the strong absorption from normal hemoglobin. This dilution lysed the cells and so also 

served to minimize light scattering. Scattering was 

further reduced by adding 20 g of a nonionic detergent, dimethyldodecylamine oxide ("Ammonyx LO"; 

Onyx Chemical Co., Jersey City, N. J.) per liter, 

which immediately dissolves the cell stroma. 

This dilution was effected by adding 20 pA of blood, 

obtained either directly from a finger puncture into a 

heparinized microhematocrit tube (Clay-Adams) or 

by means of an Eppendorf pipet from blood collected 

by venipuncture into heparinized Vacutainers (Becton-Dickinson), to 10 ml of phosphate buffer (10 

mmol/liter, pH 7.4) containing the detergent. When 

so diluted, the fluorescence of the resulting solution 

remains constant for at least a week. Even if whole 

blood is stored for a week in the refrigerator before 

being diluted with the detergent solution, the measured fluorescence of the ZPP is not significantly af-

-fected. 

The fluorescence spectrum of each diluted blood 

sample was recorded with the excitation monochrometer set at 424 nm. The intensity of the spectrum of 

594 nm (scattering background subtracted) was measured in arbitrary units, which were converted to 

concentration of ZPP (in j*g/100 ml of blood) with 

the complex of zinc protoporphyrin7 and apohemoglobin used as a standard.8

 The latter material, prepared as described by us (18), was assayed spectrophotometrically with use of the value 1.22 X 105

 for 

the absorptivity at 423 nm (20). For day-to-day referencing of fluorescence intensity data, the spectrofluorometer was calibrated with a coproporphyrin I 

standard (Sigma Chemical Co.). 

The procedure of Hessel (21), as modified by Joselow and Bogden (22), was used to determine lead in 

whole blood. 

* Zinc protoporphyrin IX may be purchased from Porphyrin 

Products (Dr. Bruce Burnham), P.O. Box 31, Logan, Utah 84321. 

s

 In order to facilitate work in other laboratories we have developed as a secondary standard zinc protoporphyrin 0.05 ng/ra\ in 

pyridine. The latter solution may be prepared as follows: Dissolve 

zinc protoporphyrin in pyridine; adjust the absorbance at 425 nm 

to 1.0/cm; dilute 100-fold with pyridine. The fluorescence spectrum of this solution is very similar to that of the lead-intoxication 

blood in dilute detergent solution. The concentration of ZPP in the 

diluted blood may be determined, by using this standard and the 

formula: 

Z P P (Mg/100 ml blood) = 

1.34 x 5 x d 

I. 

where I is the fluorescence intensity (594 nm) of the diluted blood 

sample, I, is the fluorescence intensity (at 594 nm) of the standard 

obtained under identical conditions (excitation 425 nm), 5 is the 

concentration (/ig/100 ml) of ZPP in the standard prepared as directed above, d is the blood dilution factor (500 for 20 u\ blood diluted into 10 ml buffered detergent), and 1.34 is the calibration 

factor which we have determined based on the zinc protoporphyrin-apohemoglobin standard. 

i 

10 ZO 30 40 50 60 70 60 90 

Pb (ug/IOOml b>ood) 

Fig. 2. Relation between fluorescence intensity at 594 nm of 

diluted whole blood and the whole blood lead concentration 

Data are for blood from 250 young children (small filled circles) and 35 men 

(larger open circles). The fluorescence intensity units were chosen to reflect 

(within 10%) the concentration of zinc protoporphyrin (ZPP) as /ig ZPP per/ 

100 ml of blood. The upper line is the regression line for a linear fit (r = 0.77) 

of the childrens' data: it represents the equation log ZPP Oig/100 ml) = 0.52 

+ 0.027 Pb (/jg/100 ml). The lower line is the regression line (r = 0.87) for 

the data for the adults: log ZPP (jig/100 ml) = 0.54 + 0.017 Pb (j.g/100 ml). 

(No model has been presented to justify a linear relationship between the log 

of the porphyrin fluorescence and lead concentration and we do not consider 

this semilog plot to be the most reasonable way to display the relationship. 

However, others (6, 14) have fitted similar data in this way and we do so so 

that our results may be better compared with theirs.) 

The fluorometric measurements were performed at 

Bell Laboratories, the lead determinations at the 

College of Medicine and Dentistry of New Jersey. 

The study was done in the usual double-blind manner. The specimens were received from children who 

were taking part in a screening program in the Newark, N. J. area. The specimens from men were obtained from a random group of workers at a smelting 

plant. One fluorometric test and one lead determination was made on each specimen. All the data were 

used in the statistical analysis. 

Results 

A semilog plot of fluorescence intensities vs. lead 

concentrations for 250 blood specimens from children 

(ages, 1% to 5 years) is shown in Figure 2, along with 

similar observations for 35 men occupationally exposed to lead in a smelting plant. 

The plot suggests that the log of porphyrin fluorescence at 594 nm and lead concentration are linearly 

related. This is similar to the pattern observed by 

others who have measured, at different wavelengths 

(3-7, 13-15), the porphyrins extracted by various 

techniques from blood. 

Moreover, the fluorescence associated with the 

blood specimens from the men is considerably less 

CLINICAL CHEMISTRY, Vol. 21 , No. 1. 1975 95 
P.5 

than that found in the blood of children who had 

comparable lead concentrations. In the concentration 

range of 40-60 Mg/100 ml, adult blood may show from 

% to % the porphyrin fluorescence exhibited by children's blood. 

Discussion 

Regardless of the validity or logic of the approach, 

a quantitative test intended for use in mass screening 

will require the designation of some number as a cutoff point, a limiting value below which further action 

is not indicated. 

These numbers have generally been set empirically, usually after considerable statistical manipulations that do little to enhance the strength of the diagnostic test, and which eventually involve a judgment of benefit vs. risk. The benefit to be sought is 

the widest screening coverage at the lowest possible 

cost; the risk to be avoided is the occurrence of false 

negatives. 

Table 1 shows the number of false negatives (i.e., 

children who have some degree of lead poisoning, but 

would be missed) for several blood lead concentrations and fluorescence cut-off values encountered in 

this "study of unselected specimens. 

We selected a conservative range of fluorescence 

cutoff values, because of the extreme simplicity and 

low cost of the test, an approach also warranted by 

the limitations of the test. Despite the sensitivity 

available through measurement of fluorescence, several indirect factors common to all tests that depend 

on a porphyrin measurement predispose toward inaccuracy and uncertainty: 

1. ZPP, even more than lead, is associated predominantly 

with erythrocytes. Therefore, strictly speaking, the whole 

blood fluorescence intensity should be corrected for thev 

hematocrit or hemoglobin value (3, 6), but such a correction would be significant only when more than moderate 

anemia is present. Correction of our values for the hematocrit made no significant difference with respect to a screening application. 

2. The appearance of protoporphyrin fluorescence is a 

delayed phenomenon related to erythropoiesis and occurs 

some time after the individual has been exposed to chronic 

absorption of lead (4). Thus, a child's blood may possibly 

show a high lead concentration (as a result of a very recent 

ingestion of a leaded paint chip, for example) and still be 

low in porphyrin fluorescence. 

3. An iron-deficiency anemia, as noted earlier, may also 

result in blood with a zinc porphyrin fluorescence (18). 

In an inner-city population of children, prevalence 

of iron deficiency anemia may be as high as 10%9 and 

will have some effect upon the blood lead-ZPP correlations. On this account alone, however, the ZPP test 

will also offer the additional virtue of detecting children in need of medical attention for reasons other 

than lead intoxication. It would almost be mandatory 

in a screening program to look for an iron-deficiency 

anemia among those children who show an initial 

Table 1. False Negatives Observed at Several 

Fluorescence Values" 

Lead concentration, i.g/100 ml 

Blood 240 &50 260 

ZPP.M9/ -

100 ml Proportion % Proportion % Proportion % 

15 3/250 1.2 0/250 0 0/250 0 

20 9/250 3.6 0/250 0 0/250 0 

25 14/250 5.6 2/250 0.8 0/250 0 

• Blood lead concentrations in children have become arbitrarily divided into several categories in accordance with recommendations of the Public Health Service (2i) as well as general 

practice: (a) those less than 40 pg/100 ml blood are at present 

considered acceptable or not actionable, even though undesirable; (6) those between 40 and 50pg/100 ml indicate excessive 

absorption, a cause for concern, and generally those above 50 

Mg/100 ml invoke a recommendation for immediate follow-up; (r) 

concentrations greater than 60/.g/100 ml, in many health jurisdictions, are considered as diagnostic for lead poisoning, and 

hospitalization may follow. Any blood lead poncentration >40 

fig/100 ml can thus be considered as a positive finding. 

9

 Piomelli, S., and Chisolm, J. J., Jr., personal communications. 

high ZPP value and a confirmed low blood lead concentration. 

Despite the foregoing qualifications, the simplicity 

of the ZPP test warrants its serious consideration as a 

primary screening test. If a cut-off fluorescence value 

of 15 is set, then from this study we would predict 

that all lead values greater than 50 jtg/100 ml will be 

discovered; and that less than 2% of those children 

with a blood lead concentration of 40 Mg/100 ml or 

more would be missed. On the other hand, about 25% 

of the screened children may show false positives. 

These children would be retested directly for blood 

lead. Because fluorescence can be measured quickly 

(within a few minutes after specimen collecting), 

such a retest need not involve recalling the child and 

the complications of scheduling and following 

through on a re-appointment. 

If a higher cut-off value (20 instead of 15) were 

used, a smaller proportion of false positive (about 

17%) would be observed, but a somewhat larger proportion (about 4%) of children who might be in need 

of medical attention would be missed. This might be 

considered too high a risk to take for the benefit (i.e., 

the fewer fluorescence tests) realized. 

Besides being a simpler test, the ZPP fluorescence 

assay on whole blood should be inherently more accurate than are those tests that involve an extraction 

step. No extraction procedure is used here, so the direct measurement cannot be artifactually low because of incomplete extraction. 

The ZPP test can be simplified even further by use 

of a fluorometer that is built with "front surface" optics. For the procedures described above, dilution of 

the blood was necessary only because of the right 

angle optics of the spectrofluorometers most commonly availablerWith "front surface" optics, fluorescence can be measured directly on a drop of blood on 

96 CLINICAL CHEMISTRY, Vol. 21. No. 1. 1975 
P.6 

a glass slide. A small, inexpensive portable instrument with such a design has been constructed by J. 

Eisinger and W. E. Blumberg at Bell Laboratories, 

and is currently undergoing field testing. This instrument should make it possible to examine children in 

high-risk populations often enough to substantially 

increase the pubic health benefits derived from this 

test. 

In summary: ZPP has been demonstrated to be one 

of the metabolites that accumulate in whole blood as 

a result of lead interference with heme synthesis. The 

direct measurement in whole blood of the fluorescence attributable to ZPP offers a simple, sensitive, 

specific and valid test for lead intoxication or an 

iron-deficiency anemia. Other porphyrin-related disorders, such as erythropoietic protoporphyria or porphyria cutanea tarda can be detected in a similar 

way. 

The validity and usefulness of porphyrin measurements as diagnostic criteria of undue lead absorption 

have been demonstrated by the studies of Piomelli 

(5-7), Chisolm (3), and Granick and Sassa (13, 

14) and their coworkers. The extreme simplicity and 

rapidity of our test warrants its consideration as a 

primary screening test for monitoring the effects of 

lead absorption. 

We thank S. Piomelli, J. Eisinger, and W. E. Blumberg for helpful discussions, and Velma Hinch for excellent technical assistance. W. E. Blumberg aided importantly in data handling. 

References 

1. Baloh, R. W., Laboratory diagnosis of increased lead absorption. Arch. Environ. Health 28,198 (1974). 

2. Chisolm, J. J., Screening techniques for undue lead exposure in 

children: Biological and practical considerations. J. Pediat. 79,719** 

(1D71). 

3. Chisolm, J. J., Mellits, E. D., Keil, J. E., and Barrett, M. B., A 

simple protoporphyrin assay microhematocrit procedure as a 

screening technique for increased lead absorption in young children. J. Pediat. 84,490 (1974). 

4. Chisolm, J. J., Screening for lead poisoning in children. Pediatrics 51,280 (1973). 

5. Piomelli, S., (a) Micromethod for free erythrocyte porphyrins. 

Clin. Res. 70, 497 (1972); (6) A micromethod for free erythrocyte 

porphyrins: The FEP test. J. Lab. Clin. Med. 81,932 (107:.). 

6. Piomelli, S., Davidow, B., Guinee, V., et al., The FEP (free 

erythrocyte porphyrins) test: A screening micromethod for lead 

poisoning. Pediatrics 51,254 (1973). 

7. Piomelli, S., Davidow, B., and Guinee, V. F., Lead poisoning: 

Status of the FEP and other tests. Pediatrics 52, 468 (1973). Letter to the Editor. 

8. Van Den Bergh, A. A. H., and Grotepass, W., Porphyrinamie 

ohne Porphyrinuria Klin. Wochenschr. 12, 586 (1933). 

9. Watson, R. J., Decker, E., and Lichtman, li. C, Hematologic 

studies of children with lead poisoning. Pediatrics 21,40 (1958). 

10. Vanotti, A., and Delachaux, A., Iron Metabolism and Its Clinical Significance. Grune, New York, N. Y., 1949. 

11. Schwartz, S., Berg, M. H., Bossenmaier, I., and Dinsmore, H., 

Determinations of porphyrins in biological materials. In Methods 

of Biochemical Analysis, 8, D. Glick, Ed., Interscience, New York, 

N.Y., 1960, pp 253-255. 

12. Whitaker, J. A., and Vietti, T. J., Fluorescence of the erythrocytes in lead poisoning in children: An aid to rapid diagnosis. Pediatrics 24,734 (1959). 

13. Granick, S., Sassa, S., Granick, J. L., et al., Assays for porphyrins. Proc. Nat. Acad. Sci. U. S. 69,2381 (1972). 

14. Sassa, S., Granick, J. L., Granick, S., et al., Studies in lead poisoning. Biochem. Med. 8,135 (1973). 

15. Kammholz, L. P., Thatcher, L. G., Blodgett, F. M., and Good, 

T. A., Rapid protoporphyrin quantitation for detection of lead poisoning. Pediatrics 50,625 (1972). 

16. Marver, H. S., and Schmid, R., In The Metabolic Basis of Inherited Diseases, 3rd ed., J. B. Stanbury, J. B. Wyngaarden, and 

D. S. Fredrickson, Eds. McGraw-Hill, Inc., New York, N. Y., 1972, 

pp 1087-1140. 

17. Heilmeyer, L., Disturbances in Heme Synthesis. C. C Thomas, Springfield, 111., 1966. 

18. Lamola, A. A., and Yamane, T., Zinc protoporphyrin in the erythrocytes of patients with lead intoxication and iron deficiency 

anemia. Science 186,936 (1974). 

19. Chisolm, J. J., Jr., Hastings, C. W., and Cheung, D. K. K., Microphotofluorimetric assay for protoporphyrin in acidified acetone 

extracts of whole blood. Biochem. Med. 9,113 (1974). 

20. Leonard, J. J., Yonetani, T., and Callis, J. B., A fluorescence 

study of hybrid hemoglobins containing free base zinc protoporphyrin. Biochemistry 13,1460 (1974). 

21. Hessel, D. W., A simple and rapid quantitative determination 

oflead in blood. At. Absorption Newslett. 1,55 (1968). 

22. Joselow, M. M., and Bogden, J. D., A simplified micromethod 

for collection and determination of lead in blood using a paper 

disk-in-Delves Cup technique. At. Absorption Newslett. 11, 99 

(1972). 

23. Medical aspects of childhood lead poisoning. The SurgeonGeneral's policy statement, U. S. Public Health Service. Pediatrics 

48,464(1971). 

i t * 

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CLINICALCHEMISTRY.VOL21, No. 1, 1975 97