Scandinavian Journal of Clinical and Laboratory Investigation

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Erythrocyte-Zn-protoporphyrin as an indicator of lead exposure P. Grandjean & J. Lintrup To cite this article: P. Grandjean & J. Lintrup (1978) Erythrocyte-Zn-protoporphyrin as an indicator of lead exposure, Scandinavian Journal of Clinical and Laboratory Investigation, 38:7, 669-675 To link to this article:

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Scand. J. clin. Lab. Invest. 38, 669-675, 1978.

Erythrocyte-Zn-protoporphyrin as an indicator of lead exposure

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P. G R A N D J E A N & J . L I N T R U P Institute of Hygiene, University of Copenhagen, and Department of Clinical Chemistry, Finsen Institute, Copenhagen, Denmark

Grandjean, P. & Lintrup, J. Erythrocyte-Zn-protoporphyrin as an indicator of lead exposure. Scand. J. clin. Lab. Invest. 38, 669675, 1978. Measurement of erythrocyte-Zn-protoporphyrinwith a haematofluorimeter on blood samples from eighty-four lead-exposed individuals has been compared with a fluorimetric determination of the extracted porphyrins. The results agreed well, and the interference from bilirubin and unchelated protoporphyrin was low. An exponential increase in erythrocyte protoporphyrin and a linear decrease in blood haemoglobin with increased blood lead was found in seventy-nine males with occupational lead exposure. The haematofluorimeter is recommended as a useful tool in screening for lead poisoning. Key-words: blood chemical analysis; fluorimetry ; haemoglobin ; lead poisoning; porphyrins

P . Grandiean, M .D., Environmental Sciences Laboratory, Mount Sinai School of Medicine, One Gustave Levy Place, New York, N . Y . 10029, U S A .

Traditionally, the determination of blood lead has been used as the primary method for assessing the absorption of lead [14]. The blood lead analysis, however, is subject to contamination and, in many cases, considerable analytical variation [8]. Moreover, the blood lead level reflects the recent exposure rather than the metabolically active lead [14]. Attention has therefore also been drawn to the measurement of metabolites known to be changed by lead exposure, especially haem metabolites, since lead interferes with several steps in the synthesis of haem, and lead poisoning may cause anaemia [141. Thus, analysis of 8-aminolaevulinate and coproporphyrin in the urine are in common use for the control of occupational lead expos00365-513/78/1100-0669 $02.00 0 1978 Medisinsk Fysiologisk Forenings Forlag F

we, and the measurement of aminolaevulinate dehydratase has also been recommended for this purpose [14]. Moreover, lead absorption causes an increase in the protoporphyrin content of the circulating erythrocytes [2, 91. This protoporphyrin has been shown to be predominantly chelated with zinc [6]. In iron deficiency a similar accumulation of zinc protoporphyrin (ZPP) will occur, while the increased erythrocyte protoporphyrin level in erythropoietic protoporphyria is mainly unchelated [5,6,10]. Such protoporphyria patients suffer from photosensitivity which is neither a symptom of lead poisoning nor of iron deficiency anaemia, and therefore differences in the pathogeneses of these conditions must exist [5, 10, 121. The main part of the protoporphyrin in patients with erythropoietic proto669

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P . Grandjean & J . Lintrup

porphyria is known to be loosely attached to haemoglobin [lo, 121, while ZPP is probably firmly bound to the haem binding sites [ 5 ] . Therefore, the erythrocyte ZPP will represent the average eKect of lead absorption and iron deficiency during the previous 3-4 months, i.e. the time period when the erythrocytes underwent maturation [I]. A simple, portable instrument, a haematofluorimeter, has been designed to measure ZPP rapidly in the field [I]. This instrument is a filter fluorimeter with front surface illumination. It measures the ratio between t h e ZPP fluorescence and the absorption of light by oxyhaemoglobin [l]. The prototype which was built at the Bell Laboratories in New Jersey gave results similar to determinations by other methods [I]. This article describes the performance of one of the commercially available haematofluorimeters and its use in detecting increased lead absorption.

MATERIAL Blood samples from ten healthy individuals were used for ZPP analysis with the haematofluorimeter under different conditions. An exposed population of eighty-one males and three females with different degrees of longterm occupational exposure to lead was obtained from a screening programme. Blood was drawn from the ear lobe for ZPP measurement, and venous blood was obtained from a cubital vein for other analyses. In addition, venous blood samples were collected from four males and six females with erythropoietic protoporphyria.


Zinc protoporphyrin An Aviv Haematofluorimeter Model ZPP Meter was used. A drop of blood is placed on a 25 x 25 mm2 cover glass (Corning No. I ) on the sledge of the instrument. Some cover glasses from other dealers were found to give off fluorescence which disturbed the readings. The haematofluorimeter measures the fluorescence of a standard slide of Rhodamin B, a blank slide, and the sample [I]. The result in limo1

ZPP/mol Hb (Fe) (or other unit chosen) is displayed in a few seconds. In North America, pg ZPP/g H b is a preferred unit which is approximately twenty-five times greater than the unit used in this study. Total protoporphyrin

One hundred microlitres of whole blood was diluted with 1800 p1 ethyl acetate. Proteins were subsequently precipitated by the addition of 600 pl glacial acetic acid. The separate additions of ethyl acetate and glacial acetic acid reduced the tendency to co-precipitation of porphyrins, which was further minimized by stirring during the addition of glacial acetic acid. 4 ml 1 mol/l HCI were added in order to extract the porphyrins from the organic phase. After centrifugation a sample of the clear aqueous phase which now constituted about 4.8 ml was pipetted off for scanning on a Perkin-Elmer MPF-4 fluorescence spectrophotometer. The excitation and emission wavelengths were scanned simultaneously, and the resulting fluorescence curve was analysed on the assumption that the fluorescent constituents were protoporphyrin and coproporphyrin on a nonspecific linear fluorescent background. Any uroporphyrin present would come out as coproporphyrin in this procedure. All protoporphyrin analyses were performed in duplicate, and the coefficient of variation was 5%.

For standardization protoporphyrin-IX dimethyl ester (Sigma P5880) was hydrolysed in 7.5 molil HCI subsequently diluted to 3 mol/l and kept at 4°C in the dark. The concentration was determined by absorptiometry in 1.37 mol/l HCI. For the molar absorbance of protoporphyrin in this solution we used the value of 275. lo" 1 -mol-' .cm-'. This value was found by Grinstein & Wintrobe in 1948 [3] and was adopted by Rimington in his recommendations of 1960 [ll]. In our preparation we = 1.740 which had k = 2-(A380+A430)/Amax is in reasonable agreement with the k value of 1.668 reported by Grinstein & Wintrobe 131. The absorbance spectrum of a 10 pmol/l solution remained virtually unchanged for a year. For coproporphyrin standardization we used coproporphyrin-I (Sigma COP-1-5) and 489 . lo3 1 . mol- . cm-I for the molar absorbance in 0.1 mol/l HCI as recommended by Rimington [ll].

Protoporphyriti in lead exposure



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Venous blood samples from the lead-exposed workers were drawn in lead-free Vacutainer tubes with sodium heparinate, and the determinations of blood lead were carried out by the method described by Hessel 141 at the National Institute of Occupational Hygiene in Copenhagen. Lead analysis of the blood samples from the protoporphyria patients was performed by electrothermal atomic absorption. Both methods have shown reliable results in intercomparison programmes.












Added bilirubin (urnol/l) serum


For the determination of blood haemoglobin either a Goulter S or a Hemalog 8/90 was used. Both instruments were ultimately standardized by comparison with a cyanhaemiglobin standard (Baker 3061) in a Zejss spectrophotometer.

RESULTS Factors interfering with ZPP determination

The fluorescence reading of ZPP in venous blood was found significantly lower than that of capillary blood from the ear lobe (Table I). However, when the venous blood was oxygenated by agitation with oxygen the fluorescence reading approached that of capillary blood. Repeated measurements after storage in a

FIG.1. Influence of serum-bilirubin concentration o n ZPP reading of the haematofluorimeter determined by additions of bilirubin to the same blood sample.

refrigerator for 9 weeks showed no constant trend in the results, and the average coefficient of variation was 5 %. Haemolysis induced by freezing or by the addition of a detergent resulted in unpredictable decreases in the readings (Table I). Interference from bilirubin in the serum results in high ZPP readings as demonstrated by additions of increasing amounts of bilirubin (Merck, art. 24520) to a blood sample (Fig. 1). As the upper limit of the reference interval is about 17 pmol/l serum, the interference from bilirubin would, normally, not add to the ZPP reading by more than 10 pmol/mol Hb (Fe). Likewise, the interference from fluorescent

TABLE I . Haematofluorimeter reading (prnol ZPP/rnol Hb(Fe)) of capillary vs. venous blood samples treated in ditferent ways

Patient number

Capillary blood Fresh

1 2 3

4 5 6

7 8 9 10

Mean difference from capillary blood I t l ( f = 9) P

53 62 67 69 69 79 86 88 112 121

Venous blood

__ Fresh 37

52 41 51 44 48 62 47 77 110

23.7 7.38 0.000


9 weeks old

Oxygenated haemolysed

51 69 65 66 71 16 80 84 104 130

52 65 57 66 74

33 46 29 48 31 33 74 57 71 86

1 .0 0.58 0.576


110 87 102


- 2.6 0.59 0.570

29.8 8.06 0.000

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P. Crandjean & J . Lintrup

unchelated protoporphyrin was examined. It was found that about 6% of the added amount of protoporphyrin was read as ZPP on the haematofluorimeter.

ZPP axis of Fig. 2. None of the blood samples contained significant amounts of uro- or coproporphyrin as measured by the extraction method.

Correlation between measurements of Zn protoporphyrin and total protoporphyrin

Relation of ZPP to lead and haemoglobin concentrations

The two methods for determination of protoporphyrin are closely correlated in the eighty-four subjects with occupational lead exposure (Fig. 2). The Spearman rank correlation coefficient (rs) is 0.99. However, the regression lines indicate that theextraction method for total protoporphyrin gives results which are only 82% of the haematofluorimeter readings. Repeated extraction on the same blood sample after redispersion of the protein precipitate revealed that the first extraction is 86% efficient, and the first and second extracts include 98% of the protoporphyrins in the sample. Only the result of the first extraction has been used here. If the results are corrected for this bias the slope of the regression lines will be close to unity. A small but significant intercept at 35 ymollmol Hb(Fe) appears on the

Blood lead was measured in seventy-nine of the exposed males, and the relation between ZPP and lead is shown in Fig. 3. The rs is 0.82 (P

Erythrocyte-Zn-protoporphyrin as an indicator of lead exposure.

Scandinavian Journal of Clinical and Laboratory Investigation ISSN: 0036-5513 (Print) 1502-7686 (Online) Journal homepage:
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