Researchin VeterinaryScience1992,53, 260-263

Erythrocyte protoporphyrin concentrations in clinically normal cats and cats with lead toxicity C. G. HAWKE, J. E. MADDISON*, Department of Pharmacology, The University of Sydney, New South Wales, 2006, Australia, V. POULOS, Department of Biochemistry, Royal Prince Alfred Hospital, Camperdown, New South Wales, 2050, Australia, A. D. J. WATSON?, Department of Veterinary Clinical

Sciences, The University of Sydney, New South Wales, 2006, Australia

Erythrocyte protoporphyrin (EPP) and bltood lead concentrations were determined in 91 clinically healthy cats living in the inner suburban area of Sydney, Australia. The mean EPP concentration was 223"4 _+ 186.1 gg litre -1 whole blood and the mean blood lead concentration 0"62 _4- 0-25 gmol litre -1. EPP concentrations were also monitored in three cats with confirmed lead toxicity at the time of diagnosis and one week and one month after chelation therapy with calcium EDTA. EPP concentrations were elevated in two cats and within the normal range in the third cat at the time of diagnosis. EPP concentrations were higher in two cats one week after treatment than at the time of diagnosis. One month after chelation therapy, EPP concentrations were normal in two cats but still substantially elevated in the third cat although its blood lead concentration had returned to normal and all clinical signs of lead toxicity had resolved. It was determined that the predominant form of protoporphyrin present in cats with lead toxicity was zinc protoporphyrin. The EPP assay may have limited value in the diagnosis of acute lead toxicity and in monitoring the success of chelation therapy in cats.

LEAD poisoning has been recognised in man for over 2000 years (Clarke 1973). Although lead toxicity is well documented in dogs, it is not commonly diagnosed in cats. It is generally assumed that cats have selective eating habits which reduce their risk of exposure in comparison to dogs. However, cats' grooming habits may predispose them to the ingestion of lead particles contaminating their coats. Lead has toxic effects on many cellular Components and enzymes and is described as a general protoplasmic poison (Oehme 1978). Several enzymes of the haem synthetic pathway are particularly sensitive to small quantities of lead and hence a major diagnostic focus is on this enzyme system (Kaneko 1989). Of the enzymes involved in haem synthesis, the two which *Reprint requests to Dr J. Maddison ~-Present address: Department of Medical Sciences, School of VeterinaryMedicine,UniversityofWisconsin-Madison,2015Linden Drive West, Madison,Wisconsin,53706,usa

appear to be the most sensitive to lead are ~z-aminolevulinate dehydrase and ferrochetalase (Kaneko 1989). The measurement of the accumulated substrates of these enzymes (8-aminolevulinicacid [~)--ALA])and erythrocyte protoporphyrin (EPP) is commonly used to detect lead exposure. The measurement of EPP has been widely used to screen for lead exposure in children (Piomelli et al 1973), and has been shown to be useful and sensitive in aiding diagnosis of clinical and subclinical lead toxicity in cattle (George and Duncan 1981). A positive EPP test reflects exposure to lead associated with toxic metabolic effects, notably interference with haem synthesis in the bone marrow (Piomelli et al 1973). The advantages of EPP measurement include its simplicity, rapidity and reliability (Piomelli et al 1973). The exponential increase of EPP levels compared with blood lead concentration results in the magnification of differences between normal and abnormal levels. However, increased EPP levels are not specific to lead toxicity and may also result from erythropoietic protoporphyria, iron deficiency and bilirubinaemia (George and Duncan 1981, Skerfving 1988). The aim of the present study was to establish a reference range for EPP concentrations and blood lead concentrations in cats residing in an urban environment. The urban environment was the inner suburban area of a large city with a high population and traffic density and many older houses undergoing renovation. In addition, EPPand blood lead concentrationswere determined and characterised in three cats with lead toxicity. Blood was collected from 91 cats residing in the inner suburbs of Sydney. The cats were clinically healthy and presented to veterinary practices for routine procedures such as desexing or teeth cleaning. Any cats not considered clinicallyhealthy were excluded. Two to 5 ml samples of blood were collected from each cat into plastic tubes containing lithium heparin and stored at -20°C until analysis. The mean + SD age of the clinically normal cats was 3-68 + 2-28 years (range 0.42 to 15 years). Lead toxicity was diagnosed in three cats based on appropriate clinical signs, increased blood lead con-


Erythrocyte protoporphyrins in cats centration and increased urinary ~-ALA concentration (determined in different c o m m e r c i a l laboratories utilised by referring practitioners). The clinical signs reported in three cats with confirmed lead toxicity were anorexia (three out of three), weight loss (two out of three), vomiting (one out of three), regurgitation (one out of three), abdominal pain (one out of three), lethargy (one out of three) and failure to g r o o m (one out of three). All cats h a d a normal h a e m o g r a m and routine biochemistry. Blood was collected into lithium heparin for EPP determination at the time of diagnosis and at one week and one m o n t h after chelation therapy with calcium EDTA (Calsenate; Parnell Laboratories; 100 m g kg~ld 1 subcutaneously in divided doses). All samples were stored at - 2 0 ° C until analysis within three months of collection. The EPP assay was based on a two-step extraction technique (Chisolm and Brown 1975, George and Duncan 1981) using fluorescence spectroscopy. All fluorescence measurements were made on a model M P F - 2 A Hitachi Perkin-Elmer fluorescence spectrophotometer equipped with a red-sensitive photomultiplier tube (Hitachi Perkin-Elmer). The excitation and emission slits were set at 10 nm and 16 nm, respectively. The emission wavelength was set at 609 n m and peak emission was seen at an excitation wavelength of 409 nm. Protoporphyrin IX (Porphyrin Products) was used as the standard. Blood lead concentrations were determined by graphite furnace atomic absorption spectrophotometry (G13C System 2000 Complete A u t o m a t e d Graphite Furnace System; GBC Scientific Equipment). The mean + SD for EPP concentrations in the 91 urban cats was 223.4 + 186.1 ~tg litre -1 whole blood. M e a n blood lead concentration was 0-62 _+0-25 #mol litre -1. The EPP data was not normally distributed (positively skewed), thus the median value (169.8 gg litre -1) was determined to be the most appropriate measure of central tendency in this population and the upper limit of the reference range defined as 2 SD above this value (542 gg litre-1). The blood lead data was normally distributed, therefore the upper limit of the reference range was defined as 2 SD above the mean (1-1 gmol litre t). The initial urinary ~i-ALA, EPP and blood lead concentrations for each cat with lead toxicity are shown in Table 1. All cats had increased (~-ALA and blood lead concentrations and two had increased EPP levels at the time of diagnosis. The response of each cat to chelation therapy is illustrated in Fig 1. Blood lead concentrations fell to within the normal range after chelation therapy in all the cats. All clinical signs were resolved by one m o n t h after treatment. EPP concentrations in cats 1 and 3 were higher one week after therapy than before therapy (Fig 1). In cats 1 and 2, EPP concentrations were within the n o r m a l range by one m o n t h after treatment.




Cat 1

5 ~. '® ~ =o ~ ~ ~ ~_

~ "



- Cat 2 Cat 3

4 . 3 2


1 0

Normal range |


Pre treatment

"T 5000- B ~ ~ 4000 v.c ~-~ 3ooo XZ O ~ o ~ ® o -~ 2000 S_~° ~ ' ~ 1000£ .~, ] Lu 0


1 week after treatment


Normal range


1 month after treatment

Pre treatment





1 week after treatment


1 month after treatment

FIG 1: Blood lead (A) and erythrocyte protoporphyrin (B) concentrations in three cats with lead toxicity, at the time of diagnosis and one week and one month after chelation therapy with calcium EDTA

However, in cat 3, the EPP concentration was still substantially elevated one m o n t h after therapy. After treatment, urine ~-ALA concentrations were measured in two of the three cats. In both cats, urine ~-ALA concentrations fell to less than 50 gmol l i t r ~ l by one m o n t h after treatment (data not shown). TABLE 1: Demographic data and pre-treatment diagnostic parameters in three cats with lead toxicity Cat 1

Demographic data Age (years) 14 Gender F(N) Breed DSH Diagnostic parameters Urinary (~-ALA (~mol litre-1) 103 Blood lead (gmol litre 1) 1.66

Cat 2

Cat 3

Normal range*

9 12 F(N) F(N) Chinchilla Persian

322 5.25

>500 4-84

~86.8 ~1.1




EPP (~g litre -1 w h o l e



* Mean _+2 SD of parameter value in 91 normal cats from inner suburbs of Sydney F(N) Neutered female DSH Domestic shorthair


C. G. Hawke, J. E. Maddison, V. Poulos, A. D. J. Watson

Erythrocyte protoporphyrin is bound firmly in the haem pockets of the haemoglobin molecule, and may chelate a zinc atom to become zinc protoporphyrin (Piomelli 1987). To determine whether EPP in the blood of cats with lead toxicity is free erythrocyte protoporphyrin (FEP), zinc protoporphyrin (ZPP) o r a combination of both, blood was fluorospectrophotometrically scanned and compared with standard solutions of FEP and zPP. The method was adapted from that of Lamola and Yamane (1974) and George and Duncan (1981). Protoporphyrin IX and zinc protoporphyrin (Porphyrin Products) were used as the standards. Blood samples with low, average and elevated levels Of EPP were scanned. Comparison of the emission spectra of pure FEP and zPP at excitation wavelengths of 409 nm and 420 nm and the emission spectra of blood from cats with lead toxicity indicated that the majority of the EPP present in cats with lead toxicity is in the form of zpP. To the authors' knowledge, this is the first report of the use of EPP determination to assess lead exposure in cats. The EPP concentration in humans used as the threshold for further investigation into the possibility of unacceptable lead exposure is 350 gg litre 1 whole blood (Debaun and Sox 1991). Fourteen per cent of clinically healthy cats in the present survey had EPP concentrations greater than 350 gg litre-1. However, in contrast to human medicine, the main concern in veterinary medicine is the diagnosis of clinical rather than subclinical lead toxicity. It is difficult to set an appropriate upper limit for EPP concentration in the cat. Some individual cats in the present study had relatively high EPP concentrations (up to eight times the population median) with no other clinical or laboratory evidence of lead toxicity. In addition, EPP concentrations were not necessarily elevated in cats with lead toxicity at the time of diagnosis. The finding of a normal gPP concentration in a cat with confirmed lead toxicity probably indicates lead exposure has been acute rather than chronic. EPP is formed in maturing erythrocytes in the bone marrow due to inhibition of ferrochetalase by lead (Sassa et al 1973, Piomelli 1987). Therefore EPP in the peripheral blood will not rise until sufficient erythrocytes containing an excess of EPP have been released into the circulation (Piomelli et al 1973). It follows that there will also be a delay in the response of EPP concentrations to chelation therapy, as normal erythrocytes released from the bone marrow gradually replace those with EPP in the peripheral circulation. This is illustrated by the response of cat 3 to therapy where the EPP concentration was still substantially elevated one month after chelation therapy, although the blood lead concentration (and urinary ~-ALA concentration - data not shown) had decreased to within the normal range and all signs of toxicity had resolved.

It was concluded from the present study that the predominant form of EPP in cats with lead toxicity is zPp. This finding has important implications with respect to the potential use of standard haematofluorometers in this species. The haematofluorometer is designed to assay zPP in a small drop of unprocessed blood (Blumberg et al 1977). The haematofluorometer should be suitable for assaying EPP concentrations in feline blood, as EPP was present almost entirely as ZPP in cats with lead toxicity. EPP has generally replaced urinary ~-ALA as the biological marker of undue lead exposure in human beings, primarily because it is inexpensiveand simple to perform. It is used extensively as a primary screening technique for subclinical lead toxicity (Debaun and Sox 1991). However, the detection of clinicalrather than subclinical lead toxicity is of primary concern in small animal practice, and screening for lead exposure is not usual. The EPP assay appeared to have limitations both in the detection of acute lead toxicity and the monitoring of response to chelation therapy in the small number of cases of lead toxicity reported in this study. An advantage of urinary ~-ALA in the diagnosis of lead toxicity is that urinary ~)-ALA increases in the acute stages of lead toxicity (Green et al 1978) when the animal is most likely to be presented with clinicalsigns. Further comparative studies o f EPP and urinary I~--ALA concentrations are needed to assess the relative merit of the two assays in the diagnosis of lead toxicity in cats.

Acknowledgements The authors thank Des Richardson for technical assistance and the following veterinary practices for their participation in this study: Haberfield, Balmain, Glebe, Surry Hills and Gladesville Veterinary Hospitals and the University of Sydney Veterinary Teaching Hospital. This study was supported by the National Health and Medical Research Council of Australia.

References BLUMBERG, W. E., EISINGER, J., LAMOLA, A. A. & ZUCKERMAN, D. M. ( t 977) The hematofluorometer. Clinical Chemistry 23, 270-274 CHISOLM, J. J. & BROWN, D. H. (1975) Micro-scale photofluorometric determination of free erythrocyte porphyrin (protoporphyrin X). Clinical Chemistry 21, 1669-1682 CLARKE, E. C. G. (1973) Lead poisoning in small animals. Journal of Small Animal Practice 14, I83-193 DeBAUN, M. R. & SOX, H. C. ( 1991) Setting the optimal erythrocyte protoporphyrin screening decision threshold for lead poisoning: A decision analytic approach. Pediatrics 88, 12I-131 GEORGE, J. W. & DUNCAN, J. R. (1981) Erythrocyte protoporphyrin in experimental chronic lead poisoning in calves. American Journal of Veterinary Research 42, 1630-1637 GREEN, R. A., SELBY, L. A. & ZUMWAIT, R. W. (1978) Experimental lead intoxication in dogs: a comparison of blood lead and urinary delta-aminolevulinic acid following intoxication

Erythrocyte protoporphyrins in cats and chelation therapy. Canadian Journal of Comparative Medicine 42, 205-212 KANEKO, J. J. (1989) Clinical Biochemistry of Domestic Animals. 4th edn. San Diego, Academic Press. pp235-255 LAMOLA, A. A. & YAMANE, T. (1974) Zinc protoporphyrin in the erythrocytes of patients with lead intoxication and iron deficiency anemia. Science 186, 936-938 OEHME, F. W. (1978) Mechanisms of heavy metal inorganic toxicities. In: Toxicity of Heavy Metals in the Environment. Part 1. Ed F. W. Oehme, New York, Marcel Dekker. pp69-85 PIOMELLI, S. (1987) Lead poisoning. In: Hematology of Infancy and Childhood. 3rd edn. Eds D. G. Nathan and F. A. Oski. Philadelphia, W.B. Saunders. pp389-412 PIOMELLI, S., DAVIDOW, B., GUINEE, V. F., YOUNG, P. & GAY, G. (1973) The FEP (free erythrocyte porphyrins) test: A


screening micromethod for lead poisoning. Pediatrics 51, 254259 SASSA, S., GRANICK, J. L., GRANICK, S., KAPPAS, A. & LEVERE, R. D. (1973) Studies in lead poisoning. 1. Microanalysis of erythrocyte protoporphyrin levels by spectrofluorometry in the detection of chronic lead intoxication in the subclinical range. Biochemical Medicine 8, 135-148 SKERFVING, S. (1988) Biological monitoring of exposure to inorganic lead. In: Biological Monitoring of Toxic Metals. Eds T. W. Clarkson, L. Friberg, G. F. Nordberg and P. R. Sager. New York, Plenum Press. pp169-197 Received March 16, 1992 Accepted April 27, 1992

Erythrocyte protoporphyrin concentrations in clinically normal cats and cats with lead toxicity.

Erythrocyte protoporphyrin (EPP) and blood lead concentrations were determined in 91 clinically healthy cats living in the inner suburban area of Sydn...
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