Occupational and Environmental Medicine 1997;54:241-246

6-Aminolevulinic acid dehydratase genotypemodifies four hour urinary lead excretion afteroral administration of dimercaptosuccinic acidBrian S Schwartz, Byung-Kook Lee, Walter Stewart, Pornchai Sithisarankul,Paul T Strickland, Kyu-Dong Ahn, Karl Kelsey

Department ofEnvironmental HealthSciences, Division ofOccupational HealthB S SchwartzW StewartP SithisarankulP T StricklandDepartment ofEpidemiology, JohnsHopkins School ofHygiene and PublicHealthB S SchwartzW StewartDepartment ofMedicine, JohnsHopkins School ofMedicine, BaltimoreMD, USAB S SchwartzDepartment ofPreventive Medicine,SoonchunhyangUniversity, Chonan,Republic ofKoreaB-K LeeK-D AhnOccupational HealthProgram, DepartmentofEnvironmentalHealth andDepartment ofCancerBiology, HarvardSchool ofPublicHealth, Boston, MA,USAK KelseyCorrespondence to:Dr Brian S Schwartz, JohnsHopkins University, Divisionof Occupational Health, 615North Wolfe Street, Room7041, Baltimore, MD,21205, USA.Accepted 20 November 1996

AbstractObjectives-Previous research suggeststhat binding of lead by 3-aminolevulinicacid dehydratase (ALAD) may vary byALAD genotype. This hypothesis wastested by examining whether ALAD geno-type modifies urinary lead excretion(DMSA chelatable lead) after oral admin-istration of dimercaptosuccinic acid(DMSA).Methods-57 South Korean lead batterymanufacturing workers were given5 mg/kg oral DMSA and urine was col-lected for four hours. Male workers wererandomly selected from two ALAD geno-type strata (ALAD1-1, ALADl-2) fromamong all current workers in the twoplants (n = 290). Subjects with ALADl-1(n = 38) were frequency matched withsubjects with ALADl-2 (n = 19) on dura-tion of employment in the lead industry.Blood lead, zinc protoporphyrin, andplasma aminolevulinic acid concentra-tions, as well as ALAD genotype, durationof exposure, current tobacco use, andweight were examined as predictors oreffect modifiers of levels ofDMSA chelat-able lead.Results-Blood lead concentrationsranged from 11 to 53 ug/dl, with a mean(SD) of 25 4 (10.2) ug/dl. After 5 mglkgDMSA orally, the workers excreted amean (SD) 85 4 (45.0),ug lead during afour hour urine collection (range16*5-184-1 jug). After controlling for bloodlead concentrations, duration of expo-sure, current tobacco use, and bodyweight, subjects with ALADl-2 excreted,on average, 24 Mg less lead during the fourhour urine collection than did subjectswith ALADl-l (P = 0.05). ALAD geno-type seemed to modify the relationbetween plasma 3-aminolevulinic acid(ALA) and DMSA chelatable lead.Workers with ALADl-2 excreted morelead, after being given DMSA, withincreasing plasma ALA than did workerswith ALADl-1 (P value for interaction =0.01).Conclusions-DMSA chelatable lead maypartly reflect the stores of bioavailablelead, and the current data indicate thatsubjects with ALAD1-2 have lower storesthan those with ALADl-l. These dataprovide further evidence that the ALADgenotype modifies the toxicokinetics oflead-for example, by differential bindingof current lead stores or by differences in

long term retention and deposition oflead.

(Occup Environ Med 1997;54:241-246)

Keywords: 3-aminolevulinic acid dehydratase geno-type; chelating agents; dimercaptosuccinic acid; lead

The factors that mediate the absorption, dis-tribution, and excretion of lead are complexand poorly understood. None the less, bindingof lead by specific proteins in different tissuesprobably has a major influence on the toxico-kinetics, and perhaps the toxicity, of lead.' -6Known polymorphisms in genes coding forseveral of these proteins may account for sig-nificant variation in intravascular and soft tis-sue binding and in the long term deposition oflead. One such possible polymorphism hasbeen described for the 5-aminolevulinic aciddehydratase (ALAD) gene, which codes forone of three isozymic proteins (termedALAD1-1, ALAD1-2, and ALAD2-2).7-14ALAD genotype seems to modify the toxi-

cokinetics of lead. Investigators first found dif-ferences in blood lead concentrations bygenotype, with higher concentrations in sub-jects with ALAD1-2.78 14 We previouslyreported that the prevalence of ALAD1-2 wasincreased in a battery manufacturing plantwith high exposure to lead compared withplants with lower exposures.'4 Prevalence ofALAD1-2 was also higher among workerswith longer duration of exposures.'4 Smith et alhave reported that concentrations of lead intrabecular bone were higher and those in corti-cal bone were lower in carpenters withALAD1-2, although these differences did notreach significance."' Fewer studies have evalu-ated whether ALAD genotype influenceshealth effects related to lead. Smith et alreported higher blood urea nitrogen and uricacid concentrations in workers withALAD1-2,'5 and Bellinger et al reported betterneuropsychological test performance in fiveadolescents with ALAD1-2 exposed to leadcompared with 67 adolescents with ALAD1-1exposed to lead.'3The hypotheses about altered lead binding

by ALAD raise the question whether ALADmay mediate bioavailable lead stores.Dimercaptosuccinic acid (DMSA) is a leadchelating agent given orally. Urinary leadexcretion after a dose of DMSA (DMSAchelatable lead) may be a biomarker ofbioavailable lead burden.'6 Data suggest thatthe predictors of urinary lead excretion after

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10 mg/kg DMSA orally and 1 g intravenousethylene diamine tetra-acetic acid (EDTA) aredifferent, and that earlier EDTA increasesexcretion of urinary lead when DMSA is givenlater.'6 Blood lead is a strong predictor ofEDTA chelatable lead,'6-20 whereas urinary 3-aminolevulinic acid (ALA) was reported to bethe strongest predictor of DMSA chelatablelead.'6 The DMSA may preferentially removelead from soft tissue storage sites, perhaps onlyafter conjugation with cysteine, whereasEDTA primarily removes lead from bonesites.2'-27 As ALAD may be an important siteof lead binding in many soft tissue storagepools, we thought it would be of interest tocompare DMSA chelatable lead by ALADgenotype. We report on differences in DMSAchelatable lead by genotype and associationsof this measure with other biological markersof lead exposure.

Materials and methodsSTUDY POPULATIONCurrent male employees from two lead storagebattery factories in the Republic of Korea werestudied to evaluate DMSA chelatable lead,neuropsychological function, and blood pres-sure by ALAD genotype. These employeeswere part of a larger study of 308 currentworkers in three factories who were genotypedfor ALAD."4 The current study was limited toworkers at two of the three factories, bothowned by the same company. One factorymanufactures batteries for cars and lorries(plant A) and the other manufactures largeindustrial batteries (plant B). Before the con-struction of these plants in the late 1980s, thecompany had a single plant with higher leadexposures. After completion of the new plants,the long term workers employed at the olderplant were transferred to plant B. Plant Ahired new workers.Of the 30 subjects in the two plants who

were ALAD1-2, 19 who were randomlyselected from three exposure strata (< 2 years,2-5 years, > 5 years), agreed to participate inthe current study, and provided informed con-sent. A total of 38 subjects who were randomlyselected from among the 259 subjects withALADI-1 in the two plants, agreed to partici-pate, provided informed consent, and werefrequency matched to subjects with ALAD1-2by duration of exposure. The 57 subjects whocompleted testing had a mean (SD, range) ageof 32'3 (5 9, 21-50) years and had beenemployed in the lead industry for 7-2 (3-3,1-5-13-9) years. The study protocol wasapproved by the institutional review boards ofthe Johns Hopkins School of Hygiene andPublic Health and Soonchunhyang University.

STUDY VARIABLESData collection, from 1 December 1994 to 30April 1995, included a questionnaire adminis-tered by an examiner to obtain information onjob history, medical history, current symp-toms, and history of tobacco and alcohol con-sumption; neurobehavioral testing with theWorld Health Organisation neurobehavioural

core test battery and the trails A and B tests,blood pressure measurement, blood and urinecollection for blood lead, zinc protoporphyrin(ZPP), plasma ALA, urinary ALA, and esti-mation of DMSA chelatable lead. Subjectswere given 5 mg/kg dimercaptosuccinic acid(DMSA) orally and urine was collected forfour hours. Total lead excretion in four hourswas used as a measure of DMSA chelatablelead.

ALAD GENOTYPINGA modified protocol based on the polymerasechain reaction (PCR) was used for ALADgenotyping and has been previously described.6 '4Briefly, reactions were performed sequentiallyand in duplicate on 0-5 u1 of whole blood withnested primers. Reactions were completed with1 unit of Taq polymerase (Perkin Elmer Cetus)in a buffer containing 300 ng of each primer,200 ,uM of each dNTP, 0-1% Triton X100,100 mM Tris-HCl pH 8-9, 10 pM KC1, and2-55M MgC12. Blood samples were initiallypreincubated for three cycles of three minutesat 94°C and then three minutes at 55°C. Oneunit of Taq polymerase was then added.Samples were heated for two minutes at 94"Cand incubations were continued for 35 cycles of10 s at 55°C, 30 s at 71°C, and 30 s at 94°C.The initial amplification, with 3' and 5'oligonucleotide primers (5'-AGACAGACAT-TAGCTCAGTA-3') and (5'-GGCAAAGAA-CACGTCCATTC-3'), generates a 916 basepair fragment. A second round of amplificationwith a pair of nested primers (kindly providedby J Wetmur), sequences (5'-CAGAGCT-GTTCCAACAGTGGA-3') and (5'-CCAG-CACAATGTGGGAGTGA-3'), generates an887 base pair fragment. The amplified frag-ment was cleaved at the diagnostic MSP1 site,electrophoresed through a 2% agarose gel,stained with ethidium bromide, and visualisedby fluography.

LABORATORY ANALYSESPlasma and urinary ALA were measured byhigh performance liquid chromatography(HPLC)28 with a modification of the methodof Tomokuni et al.29-3" Blood and urinary leadwere measured in duplicate by flamelessatomic absorption spectrophotometry(Hitachi-Zeeman 8100, Japan) by standardaddition methods.3' These methods for mea-surement of lead in blood and urine havereported blood lead precisions of 5%-8%, orestimated urinary lead precision of 15%.32 33Also, the laboratory participates in Koreanand Japanese government quality control pro-grammes that require regular analysis of refer-ence samples provided by the programmes.During January and February 1995, the aver-age accuracy for five reference samples was +31-%. The ZPP was measured by haematoflu-orimetry (ZP Hematofluorometer-Model206, Aviv Biomedical Inc, Lakewood, NJ,USA).

DATA ANALYSISStatistical analyses were performed with theBMDP statistical software program (BMDP

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3-Aminolevulinic acid dehydratase genotype modifies four hour urinary lead excretion after oral administration of dimercaptosuccinic acid

Table 1 Summary statistics of selected study variables by 3-aminolevulinic aciddehydratase genotype in 57 Korean lead workers

ALAD genotype

Characteristic ALAD1-1 (n = 38) ALAD1-2 (n = 19) P value

Age (y) (mean (SD)) 32-1 (5 9) 32-8 (6-1) 0-68Duration of exposure (y)

(mean (SD)) 7-3 (3-3) 7 0 (3-3) 0-76Weight (kg) (mean (SD)) 62-8 (11-5) 65-7 (7-0) 0-32Employed in plant A (%) 71-1 78-9 0-52Current cigarette smokers (%) 71-1 78-9 0-52Past cigarette smokers (%) 10-5 10-5 1 0Current alcohol users (%) 76-3 78-9 0-82Blood lead in early 1995 (ug/dl)

(mean (SD)) 26-1 (9 8) 24-0 (11-3) 0-48Blood lead in late 1994 (ug/dl)

(mean (SD)) 38-5 (10-7) 34-5 (13-6) 0-23ZPP in early 1995 (ug/dl)

(mean (SD)) 57-1 (26 8) 48-3 (22-2) 0-23ZPP in late 1994 (ug/dl)

(mean (SD)) 68-9 (31 9) 55-6 (28-8) 0-13Urinary ALA in late 1994

(ng/ml) (mean (SD)) 1395 (984) 1022 (679) 0-15Plasma ALA in late 1994

(ng/ml) (mean (SD)) 17-3 (9-2) 11-8 (3 3) 0-03Four hour urinary lead excretion

after 5 mg/kg oral DMSA(ug) (mean (SD)) 92-9 (45-1) 70 3 (42-1) 0-07

P values are from t tests comparing the means in the two ALAD genotype groups.

Statistical Software, Los Angeles, CA, USA).Detailed frequency distributions and summarystatistics were examined for all study variables.Relations between variables were assessed bycorrelations, analysis of variance, and multiplelinear regression.

Linear regression was used to determinepredictors of four hour urinary excretion after5 mg/kg DMSA orally. The ALAD genotypewas evaluated as both a main effect on DMSAchelatable lead levels, and as an effect modifierof the relation between blood lead, ZPP,plasma ALA, urinary ALA, and duration of

exposure and DMSA chelatable lead. Onlyinteractions between blood lead, ZPP, plasmaALA, urinary ALA, and duration of exposureand AIAD genotype were examined; theseinteractions were identified originally as beingof interest because of previous research andhypotheses on binding of lead by ALADwithin erythrocytes.' '6 Non-linear relationsbetween the various lead biomarkers-forexample, blood lead, ZPP, plasma ALA-andDMSA chelatable lead were evaluated byinclusion of quadratic terms for the biomark-ers in the linear regression models. All linearregression models were assessed for departuresfrom normality, multicollinearity, and het-eroscedasticity.

ResultsBlood lead and ZPP concentrations suggestedthat lead exposures in these plants were mod-erate (table 1). Blood lead concentrationsranged from 11 to 53 ug/dl, with a mean (SD)of 25-4 (102) ug/dl. After 5 mg/kg DMSAorally, the 57 workers excreted a mean (SD,range) of 85-4 (45 0, 16 5-1841) yug of leadduring a four hour urine collection.

There were no differences by ALAD geno-type in age, duration of exposure, proportionof workers employed in plant A, cigarette oralcohol use, blood lead concentrations, or ZPPconcentrations (table 1). There were differ-ences in plasma ALA (P = 0-01) and concen-trations of DMSA chelatable lead (P = 0 07)by ALAD genotype (figure). The DMSAchelatable lead was correlated with blood lead

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Blood lead, DMSA chelatable lead, andplasma ALA concentrations byALAD genotype in 57 Korean male lead workers. P values are for differences inthe three biomarkers byALAD genotype. The adjusted P value for DMSA chelatable lead is after controlfor concentrations of blood lead, duration ofexposure, weight, and current cigarette use.

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Table 2 Pearson's correlations for selected study variables in 57 Korean lead workers

Pearson's r correlation coefficient*

Blood Duration of Plasma UrinaryVariable** Chellead lead ZPP exposure ALA ALA

Chellead (ug)tBlood lead in early

1995 (ug/dl) 0 40 -ZPP in early 1995

(ug/dl) 0 10 0 55 -Duration of exposure

(y) 0-18 0 10 0 14 -Plasma ALA in late

1994 (ng/dl)4 0-35 0-35 0-21 0-51Urinary ALA in late

1994 (ng/dl) -0 12 0-18 0 04 0-21 0-06

*Pearson's r values > 0 35 are associated with P values < 001. No other r values have associatedP values < 0 05.tChellead = four hour urinary lead excretion after 5 mg/kg DMSA orally.tAll correlations with plasma ALA are after elimination of three extreme values (outliers) for thisvariable.

(r = 040) and plasma ALA (r = 0 35) con-centrations (table 2). As expected, blood leadconcentrations were correlated with ZPP (r =0 55) and plasma ALA (r = 0 35) concentra-tions (table 2).ALAD genotype was an independent pre-

dictor of DMSA chelatable lead, after controlfor blood lead concentrations, duration ofexposure, current tobacco use, and weight(table 3, model 1). Subjects with ALADI1-2excreted a mean of 24 ug less lead during thefour hour urine collection than did subjectswith ALAD1-1 (P = 005). Similar relationswere found when ZPP and plasma ALA weresubstituted for blood lead in this model (table3, models 2a and 3). The ALAD genotype didnot modify the relations between blood lead orZPP concentrations and DMSA chelatablelead. In a separate model, genotype did seem,however, to modify the relation betweenplasma ALA and DMSA chelatable lead.Workers with ALAD1-2 excreted more lead,after DMSA, with increasing plasma ALA (alarger regression coefficient for the relationbetween plasma ALA and DMSA chelatablelead, table 3, model 2b) than did workers withALAD1-1 (P value for interaction < 001).Some caution should be expressed as three

people with ALAD1-1 and high plasma ALAconcentrations had a strong influence on thisrelation.

Although there were some differences inDMSA chelatable lead (P = 007) and dura-tion of exposure (P = 0 01) by month of test-ing, there were no such differences in bloodlead, ZPP, urinary ALA, or plasma ALA con-centrations. Month of testing was not associ-ated with DMSA chelatable lead in linearregression models after adjustment for bloodlead, duration of exposure, ALAD genotype,weight, and current smoking status.

DiscussionIn male lead workers with similar blood leadconcentrations, duration of exposures, andages, the data suggested that ALAD genotypeinfluenced urinary lead excretion after a5 mg/kg oral dose of DMSA. Subjects withALAD 1-2 excreted significantly less lead thandid subjects with ALAD1-1. If urinary leadexcretion after DMSA is conceptualised as asurrogate for bioavailable lead stores, then thedata imply that subjects with ALAD1-2 havelower current bioavailable lead stores. Thesedata provide further evidence that ALADgenotype modifies the toxicokinetics of lead,although the nature of the interaction betweenthe different variants of this protein and leadseems to be complex. For example, ALAD 1-2could influence DMSA chelatable lead by dif-ferential current binding of lead in theintravascular space, by modifying the longterm retention and deposition of lead, or byacting as a selection factor in the workplace(subjects with ALAD1-1 end employmentearlier, subjects with ALAD 1-2 select formore highly exposed areas) by an as yetunrecognised mechanism independent ofthese two.

There is supporting evidence for all of thesepotential mechanisms. ALAD genotype influ-ences plasma ALA concentrations, which sup-

Table 3 Linear regression results modellingfour hour urinary lead excretion after 5 mglkg DMSA orally (four hour DMSAchelatable lead)

Units of /3 /3 coefficient (95% CI) P value Model r?

Model 1 (with blood lead):Blood lead, ,ug/dl usg/tLg/dlALAD genotype (1-2 v 1-1) figDuration of exposure (y) fg/ySmoker (current v not-current) ugWeight (kg) fg/kg

Model 2a (with plasma ALA):Plasma ALA (ng/ml) uig/ng/mlALAD genotype (1-2 v 1-1) figDuration of exposure (y) fig/ySmoker (current v not-current) lugWeight (kg) fig/kg

Model 2b (effect modification by ALAD with plasma ALA):Plasma ALA (ng/ml) fug/ng/mlALAD genotype (1-2 v 1-1) fugDuration of exposure (y) fug/ySmoker (current v not-current) AWeight (kg) fug/kgCross product term (ALAD genotype fug/ng/ml

and plasma ALA, ng/ml)Model 3 (with ZPP):ZPP (ag/dl) fig/jig/dlALAD genotype (1-2 v 1-1) fugDuration of exposure (y) fg/ySmoker (current v not-current) P9Weight (kg) fuglkg

1-510 (0-442 to 2 578)23-761 (-46-67 to - 0 854)- 2-105 ( 5-433 to 1-223)19-270 ( 6026 to 44-57)0 735 (- 0-302 to 1-772)

- 0- 188 ( 1719 to 1-343)29-048 ( 54-50 to - 3-592)- 1-321 (-4890 to 2 248)27-485 (1-045 to 53-93)0-864 (-0-281 to 2-009)

- 0-653 (- 2-094 to 0 788)133-58 (- 205-4 to - 61-74)- 1-597 (- 4-882 to 1-688)25-529 (1-842 to 49 22)0-972 (- 0-082 to 2-026)8-649 (3 030 to 14-27)

0-324 ( 1-133 to 0-781)26-171 ( 50-25 to - 2-090)- 1-500 ( 4-969 to 1-969)32-494 (5-593 to 59 40)0-965 (-0-125 to 2 055)

Three separate main models were fitted, each with a different primary biomarker of lead exposure (blood lead, ZPP, and plasmaALA), because these biomarkers lie along the same casual pathway of lead exposure and the haeme synthetic system.

Variable0 30

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3-Aminolevulinic acid dehydratase genotype modifiesfour hour urinary lead excretion after oral administration of dimercaptosuccinic acid

ports the current binding hypothesis. Subjectswith AIAD1-2 have lower ALAP concentra-tions after controlling for age and blood leadconcentrations.28 Earlier studies also suggestthat subjects with ALAD1-2 have lower con-centrations of lead in cortical bone (adjustedfor age, P = 0-17), higher concentrations intrabecular bone (adjusted for age, P = 0-22),a larger trabecular cortical bone lead differ-ence (adjusted for age, P = 0-06), higher uricacid concentrations (adjusted for blood lead,age, and alcohol consumption, P = 0 09), andhigher blood urea nitrogen concentrations(adjusted for blood lead, age, and alcohol con-sumption, P = 006).'5 These data suggestthat ALAD genotype may influence leadexcretion and hence long term deposition oflead in important in vivo storage sites.'5Finally, the prevalence of AIAD1-2 has beenreported to be higher in lead plants with highexposures and among workers with longerdurations of exposure, evidence that ALADgenotype may be a selection factor in work-places that contain lead.'4 It is possible, how-ever, that differences in current binding orlong term deposition of lead may explain theseassociations with plant and duration of expo-sure.

In an earlier study of DMSA chelatablelead, Lee et al reported that blood lead was notassociated with eight hour urinary lead excre-tion after 10 mg/kg DMSA orally but that uri-nary ALA was correlated with this measure.'6In the current study, the dose of DMSA wasless (5 mg/kg) and the urine collection timewas shorter (four hours). We chose this doseand collection time to determine the predic-tors of a measure of low dose short durationDMSA chelatable lead, a protocol which isless expensive and time consuming. The lowdose four hour measure was correlated withcurrent blood lead and plasma ALA concen-trations, but was not correlated with urinaryALA concentrations, by contrast with thehigher dose eight-hour measure. It is possiblethat DMSA primarily accesses blood leadstores at low doses and blood lead and soft tis-sue stores at higher doses.

It is interesting to note that DMSA seems topreferentially remove lead from soft tissue sitesthat are rich in ALAD. Early research in sev-eral species of animals determined that ALADactivity is high in liver, kidney, and bone mar-row, and lower but still present in blood.34Although there are few comparable data inhumans, this organ and tissue distribution ofALAD is likely to be at least similar to that inpeople. It is not known whether ALAD bind-ing sites of lead in soft tissues represent a pri-mary or even majority source of lead that issubsequently bound by DMSA.

Associations of plasma ALA with AIADgenotype from this population have been pre-viously reported.28 In the current study,plasma ALA was correlated with DMSAchelatable lead and ALAD genotype seemedto modify that relation. Workers withALAD1-2 excreted more lead after DMSA foreach ng/ml plasma ALA than did workers withALAD1-1. Although this association was

heavily influenced by three subjects withALAD1- who had high plasma ALA concen-trations, it was still present after elimination ofthese subjects but did not reach significance.This association is also consistent with ourhypotheses about lead binding by AIAD.These data suggest that, in subjects withALAD1-2, the lead storage compartmentsthat influence ALAD activity (and thus plasmaALA concentrations) and from which lead ischelated by DMSA are more similar than insubjects with ALAD1-1.The increasing number of publications on

ALAD genotype now provides significant evi-dence that ALAD may be an important effectmodifier of the toxicokinetics of lead. Subjectswith ALAD1-2 have higher concentrations ofblood lead, lower concentrations of DMSAchelatable lead, lower concentrations ofplasma ALA, and possibly lower concentra-tions of lead in cortical bone and higher in tra-becular bone. In the only published studies ofdifferences in health effects by genotype,Bellinger et al reported that five subjects withALAD1-2 had consistently better neuropsy-chological test performance than did 67 sub-jects with ALAD1-1,"3 but Smith et alreported higher blood urea nitrogen concen-trations in subjects with ALAD1-2.'5 Moredefinitive evidence about possible differentialtoxicity by genotype awaits further study.

This research was supported, in part, by ES03819 (BSS),ES00002 (KK), and P42 ES05947 (KK).

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Vancouver styleAll manuscripts submitted to Occup EnvironMed should conform to the uniformrequirements for manuscripts submitted tobiomedical journals (known as theVancouver style.)

Occup Environ Med, together with manyother international biomedical journals, hasagreed to accept articles prepared in accor-dance with the Vancouver style. The style(described in full in the BM7, 24 February1979, p 532) is intended to standardiserequirements for authors.

References should be numbered consec-utively in the order in which they are firstmentioned in the text by Arabic numeralsabove the line on each occasion the refer-ence is cited (Manson' confirmed otherreports2-5 . . .). In future references topapers submitted to Occup Environ Med

should include: the names of all authors ifthere are seven or less or, if there are more,the first six followed by et al; the title ofjournal articles or book chapters; the titlesof journals abbreviated according to thestyle of Index Medicus; and the first and finalpage numbers of the article or chapter.Titles not in Index Medicus should be givenin full.

Examples of common forms of refer-ences are:

1 International Steering Committee of Medical Editors,Uniform requirements for manuscripts submitted tobiomedical journals. BMY 1979;1:532-5.

2 Soter NA, Wasserman SI, Austen KF. Cold urticaria:release into the circulation of histamine and eosino-phil chemotactic factor of anaphylaxis during coldchallenge. N EnglJ Med 1976;294:687-90.

3 Weinstein L, Swartz MN. Pathogenic properties ofinvading micro-organisms. In: Sodeman WA Jr,Sodeman WA, eds. Pathologic physiology, mechanismsof disease. Philadelphia: W B Saunders, 1974:457-72.

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