United States .
            Environmental Protection
Office of Water
December 1997
      &EPA  Information Pertaining to
            Lead in Drinking Water
            at Transient Non-
            Community Water


    "Information Pertaining to Lead in Drinking Water at Transient Non-
                          Community Water System's"
                                 EPA 815-B-97-003
                                ',   December 1997
This Publication Contains the Following Documents:

Agency for Toxic Substances and Disease Registry (ATSDR).  1992.  Toxicological Profile for
Lead. U.S. Department of Health & Human Services, Public Health Services, Atlanta, GA. TP-
92/12, pages 9, 65, 66, 98,, and 99.  ;

Giridhar, J and Isom, G:E. 1990. Interaction of Lead Acetate with Atrial Natriuretic Factor hi
Rats. Life Science, 46(8):569-576.  ,

Hindmarsh, J.T. 1986. The Porphyrias: Recent Advances. Clinical Chemistry, 32(7): 1255-
1263.                           i

Karmakar, N., Saxena, R., and Anand, S.  1986. Histopathological Changes Induced hi Rat
Tissues by Oral Intake of Lead Acetate. Environmental Research, 41(l):23-28.

Khan, A.J., Patel, U., Rafeeq, M., Myerson, A., Kumar, K., and Evans, H.E. January 1983.
Reversible Acute Renal Failure in Lead Poisoning. Journal of Pediatrics, 102(1):147-149.
Nakhoul, F., Kayne, L.E., Brautbar,N., Hu,N., McDonough, A., Eggena, P., Golub, M.S.,
Berger, M., Chang, C., Jamgotchian, N., and Lee, D.B.N.  1992. Rapid Hypertensinogenic
Effect of Lead: Studies in the Spontaneously Hypertensive Rat.  Toxicol In. Health, 8(l-2):89-

U.S. Environmental Protection Agency. December 1995. A Survey of Lead in Drinking Water
Supplied By Transient Water Systems. Prepared by Richard P. Maas, Steven C. Patch, Diane M.
Morgan, Geoffrey M. Brown. Environmental Quality Institute, The University of North Carolina
atAshville. Technical Report #95-019, December 1995.

U.S. Environmental Protection Agency. March 20,1996. Note from Jeff Cohen to Judy
Lebowich and Connie Bosma entitled: Analysis of UNC-Ashville Survey of Lead at Transient
Systems.                                                                           •

U.S. Environmental Protection Agency. November 3,1997. Effects From Short-Term Lead
Exposure. Health and Ecological Criteria Division, Office of Science and Technology, Office of
Water.                          :


Public Health Service         ;
Agency for Toxic Substances and Disease Registry
Printed on Recycled Paper
 Federal Recycling Program

                                 2.  HEALTH EFFECTS

The primary purpose of this
interested individuals and groups with at
                                                     health effects.  It  contains descriptions and
epidemiological investigations.

 sites, the information fa this section is organized Bra : »y
 L'then °y
                                                                    developmental,  reproductive,
                                                                    three iposure periods-acute

                                                              OI more)'
       Of Sircar. — *r each -teand^
     points fa the figures *omne no-observcleKe
       levels  (LOAEU) reflect

                                                                 are intended ,0 help the users of
     Si£n,ficance of the exposure £* ^^4^^

  project managers concerned ™tha^ropnaeacnons to tat            ^ ^^     Am_) or

  on levels of exposure assoctated „'"«' ™°'^°° '^een observed.  Estimates of levels postng minimal
                                    SEW e of interest to health professionais and citizens a,,ke.
  Uvels of exposure associated with the carcinogenic effects of lead are indicated fa Figure 2-2.

            of exposure lev., posmfaima, ,*»
       ou   methods have heen established
   uncertainties are associated  with  these
   uncertainties inherent in the appljcanon  of the ££*£> >™         that are delayed m development
   example,  acute inhalanon MRU may not ^P™"^™ hvpersensitivity reactions, asthma, or chronic


                                           2. HEALTH EFFECTS
  An increase in systolic blood pressure was observed in rats at a very low exposure level (1 ppm lead in
  the drinking water for 159 days, but  the dietary and drinking water content of essential and nonessential
  metals was abnormally low, and the low-contamination quarters in which the rats were housed also limited
  their exposure to  essential and nonessential metals (Perry  and  Erlanger 1978).  These conditions, which
  result in greater absorption of lead and effects  at lower lead intakes than when the diet  is less restricted
  and  the living quarters less isolated, may not be relevant to human exposure. Increases in blood pressure
  at low exposure levels have been demonstrated  in  other studies.  For example, rats administered 0.1 and
  1.0 ppm lead acetate  in  drinking water beginning at weaning through  18 months  of age exhibited  an
  approximate  14-mmHg elevation in  blood pressure from  3 months  (1  ppm)  or 12 months (0.1  ppm)
  through the entire 18 months of exposure  (Perry et al. 1988). No obvious signs of toxicity were  observed
  in these animals.

  Low-level,  chronic-duration exposure of rats to lead (30  ppm lead  acetate in the drinking water) for
  18 months resulted in a 10-15-mmHg increase  in  both systolic and diastolic blood pressure without any
  change in heart rate or histopathological evidence of damage to  the  kidney, heart, brain, aorta, or  liver
  (Carmignani et al. 1988a).  However, the  authors  were able to demonstrate that lead exposure in  these
  animals increased  their responsiveness to stimulation of a  and &  receptors,  altered the renin-angiotensin
  system,  perhaps through  inhibition of  the renin-angiotensin converting enzyme, and altered the cyclic
,  adenosine  monophosphate (cAMP)-dependent  contractile  processes  in  both myocardium and  vascular
  myocells.  Please refer to Section 2.4 for a more  complete discussion of the proposed mechanisms  for lead-
  induced hypertension.

  Cardiovascular effects other than effects on blood pressure  have also been observed in laboratory animals
  following ingestion of lead.   Male rats given 1% (10,000 ppm)  lead acetate in  their drinking water from
 6 to  12 weeks  of age had changes in the myocardium (including  myofibrillar fragmentation and separation
 with  edema fluid),  dilation of  the sarcoplasmic reticulum, and mitochondrial swelling (Asokan 1974).  Blood
 lead  levels  in these rats averaged 112  ^g/dL, versus 5 /ig/dL in controls.  The highest NOAEL values and
 all reliable LOAEL values for each study for cardiovascular effects are recorded in Table 2-4  and plotted
 in Figure 2-2.

 Gastrointestinal Effects. No studies were located regarding gastrointestinal effects in humans or animals
 after oral exposure to  inorganic lead.  See Section  for a discussion of the gastrointestinal effects
 of lead in humans after multi-route exposure.

 Hematological Effects.  As discussed in Section, lead  has  long been known  to affect heme
 biosynthesis by affecting  the activities of several  enzymes in  the heme  biosynthetic  pathway.   The
 mechanisms for these effects are discussed in detail  in Section 2.4.  Two experimental studies of the effects
 of oral exposure to lead on heme synthesis  in humans were  available. Two groups of five women and one
 group of five men who ingested lead acetate at 0.02 mg lead/kg/day  every  day for 21 days experienced
 decreases in erythrocyte ALAD by day 3 of lead ingestion  (Stuik  1974).  The decreases became  maximal
 by day" 14 and then remained constant through  day 21.  An increase  in EP occurred  in the women, but
 not in the men, starting after  2 weeks  of ingestion.  Blood lead levels were approximately 15 ^ig/dL before
 exposure and increased to approximately 40 ptg/dL during exposure.  Increased EP was observed in  five  men
 at a  higher dosage, 0.03 mg lead/kg/day (which  produced a mean  blood lead level of 46  ^g/dL), starting
 after 2 weeks  of lead ingestion (Stuik 1974).   Similar results were reported by Cools et al.  (1976) for
 11 men ingesting lead acetate at an initial dosage of 0.03 mg lead/kg/day, which  was decreased to 0.02 mg
 lead/kg/day or  less  as necessary to maintain a blood lead level of 40 ^g/dL; the  mean pre:exposure blood
 lead  level was  17.2


                                                         2.  HEALTH EFFECTS
                Intermediate-duration studies in  animals indicate that adverse  hematological  effects  (i.e.,  decreased
                hematocrit, impaired heme synthesis) occur following oral exposure (Krasovskii et al. 1979; Overmann 1977;
                Walsh  and Ryden 1984).  The lowest dose at which these effects are seen depends on the  route  of
                exposure and the nature of the  end point studied.  For example, decreases  in hematocrit were  observed
                in  rats that received 19.2 mg lead/kg/day  by gavage (Overmann 1977), but this effect was not seen until
                a dose of 318 mg lead/kg/day was administered to rats in their daily diet (Walsh and Ryden 1984).  Rats
                that received up to 34 mg lead/kg/day as lead acetate in their drinking water exhibited no adverse effects
                on hematocrit (Fowler et al. 1980;  Victery et al. 1982b). However, evidence of impaired  heme synthesis
                (increased urinary ALA and coprophobilinogen) was observed in rats that received 0.01 mg lead/kg/day as
                lead acetate in  their drinking water (Krasovskii et al. 1979).  A similar correlation between  exposure to
                lead in the drinking water as lead acetate and increased urinary ALA was observed by Fowler et al. (1980).
                Increased urinary ALA  and erythrocyte ZPP  were also correlated with increasing doses  of  lead in rats
                receiving 0, 1.67, or  6.35 mg lead/kg/day in their drinking water (Cory-Slechta 1990b).  The increase was
                observed earlier in the course of the study in older rats.

                Adverse hematological effects have  been noted in  chronic-duration studies, as well.  The effects appear to
                increase in severity with increasing dose.  For example, dose  (blood  lead)-effect information for  heme
                synthesis and hematological effects  is available; rats and  dogs were fed lead acetate in the diet for 2 years
                (Azar et al. 1973).  In  rats, lead produced  no effects at 10 ppm  (blood lead  level =  11.0 /zg/dL;  not
                elevated above  controls), significant inhibition  of ALAD at a50 ppm (blood  lead level  a!8.5 /zg/dL),
                significant increase in urinary ALA at a500 ppm (blood  lead level  a77.8 pg/dL),  and slight but significant
                decreases in hemoglobin concentration and hematocrit at a 1,000 ppm (blood lead  level ^98.6 ptg/dL).  In
                dogs, lead produced  no  effects at s50 ppm (blood lead level S31.5 /ig/dL), significant inhibition of ALAD
                at  alOO ppm (blood  lead level &42.S /zg/dL), and no effect on urinary ALA, hemoglobin, or hematocrit at
                'any exposure level  (highest exposure level  = 500 ppm, blood lead level = 75.8 fj.g/dL). Control blood lead
                levels  were 12.7 and 16.4 /jg/dL in  the two rat groups and  15.8 /ig/dL in the dogs.

                Studies in animals  indicate that the effects of lead  on heme synthesis occur in many tissues.  Oral exposure
                of rats to lead  decreased liver  ALAS activity  (Silbergeld et al.  1982), increased spleen ALAS activity
                (Silbergeld 'et al. 1982),  decreased kidney  ALAS activity (Fowler et al. 1980), decreased brain (Gerber et
                al. 1978), liver,  and  spleen  (Silbergeld et al.  1982) ALAD activity, and decreased kidney ferrochelatase
                activity along with mitochondrial injury and  disturbance of mitochondrial function (Fowler et al. 1980).
r.           ,    The highest NOAEL values and all reliable  LOAEL values for each study  for  hematological effects are
I               recorded in Table  2-4 and plotted  in Figure  2-2.

                Musculoskeletal Effects.   Several case  reports  of individuals who experienced high exposures to lead
                either occupationally or through the consumption of illicit whiskey described the occurrence of a bluish-
                tinged line  in the gums  (Eskew et al. 1961; Pagliuca et al. 1990).  The etiology of this  "lead line" has- not
                been elucidated. Individuals having high exposures to lead have also been reported to complain of muscle
                weakness, cramps,  and joint pain (Holness and Nethercott 1988; Marino  et  al.  1989;  Matte et al. 1989;
                Pagliuca et al. 1990).

                No studies were located regarding musculoskeletal effects in animals after oral exposure to inorganic lead.
                Hepatic  Effects.  No studies were located  regarding hepatic  effects  in humans  after oral  exposure to
                inorganic lead.   See Section for a discussion of hepatic effects in humans following multi-route
                exposure to inorganic lead.


                                         2. HEALTH EFFECTS
from the respiratory tract may be the lack of lead found at autopsy in the lung tissues of occupationally
exposed lead workers (Barry 1975) and  nonoccupationally exposed subjects (Gross et al. 1975).

Organic Lead.  Following a single exposure to vapors of tetraalkyl lead compounds (approximately 1 mgto3
breathed through a mouthpiece, 10-40 breaths  of approximately 1 L volume) m four male subjects, 37%
«d 51% of inhaled tetraethyl  and tetramethyl lead, respectively, were initially found in the respiratory
tract but a considerable percentage of these volatile compounds was lost through exhalation (Heard et al.
19791   Approximately 60-80% of the  deposited tetraalkyl lead was absorbed by the  lungs.  In a case
report  of a 22-year-old male  exposed  to  tetramethyl lead, absorption was  evident because  of  elevated
urinary lead levels for 4 days after exposure (Gething 1975).

Limited experimental data suggest that inhaled lead  is absorbed rapidly by animals (EPA 1986a). One hour
af£ femT Wstar rats breaThed  total lead concentrations of 0.01 mg lead/m* as tetraethyl lead m the
form of aerosolized leaded gasoline labeled with lead-210 (»°Pb) tracer for 30-45 minutes, lead c earance
 n the lungs was 30%; the majority of  the particles were 0.1-0.5 Mm in diaraeter (Boudene et a. 1977£
Immediately after nose-only breathing,  of engine exhaust  aerosols containing 6 mg lead/m* as  lead-203
SbVlabeled tetraethyl lead  for 40 or 60 minutes, 25% of  the dose was accounted for m tissues other
 ban the lung and gastrointestinal tract in rats (Morgan and Holmes 1978).   Initially  the lead content in
 ungs dec eased quite rapidly;  only 7.5% of the dose was retained in the lungs after  48 hours  followed by
a sl?wer decline in which less than 2%  of the dose remained  in the lungs after a week  The lung had.  he
SoweTtissue lead content in  rats and  rhesus  monkeys who  inhaled 0.0215  mg lead/m3 continuously (22
 hours  per day) for a year (Griffin et al.  1975b).  Oral  Exposure

 Although there were limited data, oral  absorption of lead appears to be low in humans except in children.
 The extent and rate of gastrointestinal  absorption  are affected by fasting and the solubility  of a particular
 ?ad 'alt in gastSc  acil   Male  subjects were fed  diets supplemented with 0.0008-0.003 mg/kgAIay^of
 ead-M4 (2°4)-labeled  lead nitrate for up to  124  days in which  6.5-10.9% of the tracer was absorbed
 fRabinowitz et al. 1976).  Absorption can be as high as 45% in adults under fasting conditions  to as low
  f6% S food (Chamberlain cTaL 1978).  It was reported that intestinal absorption of|touI cMonde^was
 3% with  meals but increased to 60% when  the subjects were fasted  (Heard and  Chamberlain  1982)
 LtSSvSS variability of oral absorption is shown in the study by Heusler-Bitschy et al. (988) m winch
 intestinal absorption ranged from 10% to 80% in eight  fasted volunteers receiving a single exposure of
 0.007 or 0.02  mg lead/kg/day in drinking water. .

 There is indirect evidence from autopsies which reveal high lead content in tissues, occurring from life-time
 exposure (Barry 1975).  Oral intake of lead'can result from consuming lead-containing food and beverages
 and from swallowing lead deposited in the upper respiratory tract after inhalation exposure (Kehoe 1987) -
  In addition, the ingestion of lead in children may  occur through normal mouthing activity and pica,^ which
  is the ingestion of material not fit for food, such  as soil, clay, ashes, paint chips, or plaster  (EPA 1986a).
  The primary site of lead absorption in children is the gastrointestinal tract (Hammond 1982).  For dietary
  lead   absorption in children  is approximately 50% compared with 15% gastrointestinal lead absorption
  measured in  adults (Chamberlain et  al. 1978).  When daily intake was greater than  0.005 mg lead/kg
  absorption was increased from 26.2% to 41.5% in  infants, 2 weeks to 2 years  of  age, who  were exposed
  for 72 hours to lead in milk and commercially prepared strained food (Ziegler et al.  1978).  However, the
  authors indicated that  at lower lead levels there  was difficulty  in controlling lead  intake.


                                         2. HEALTH EFFECTS
There is evidence to suggest that  gastrointestinal  absorption  in  animals  is controlled  by a saturation
phenomenon, as well as influenced by fasting (Aungst et al. 1981). In fasted rats, absorption was estimated
at 42% and 2% following single oral administration of 1 and 100 mg lead/kg, respectively, as lead acetate
fAunest et al  1981).  In mice, absorption was  14% in  fasted mice versus 7.5% in fed mice 4 hours
following an oral gavage dose of 0.003 mg lead/kg as lead acetate  (Garber and Wei 1974).  However, no
difference in  absorption (4-5%) was observed in  fasted and nonfasted mice receiving 2 mg lead/kg.

The extent of gastrointestinal absorption of lead  in experimental animals is age dependent. The rat pup
absorbs  40-50  times more  lead via the diet than does the adult rat (Forbes and Rema 1972; Kostial et
al  1978).   In  rats  receiving an  oral  dose of  1  mL lead-212 (212Pb)-labeled tracer,  absorption was
approximately 74-89% for animals  16-22 days  of age, 15-42% in  animals  24-32 days old, and only  16 /«
at 89 days old  (Forbes and Reina 1972).  A single  dose of lead resulted in 52% absorption in 1-2-week-
old suckling rats compared to 0.4% in adults (Kostial et al. 1978).  Age differences in absorption rate  were
evident  in rat pups who  had slightly higher tissue levels compared to adult rats following a single gavage
dose of 1 or 1CI mg lead/kg as lead acetate (Aungst et al. 1981).  Absorption was 37.9% for young monkeys
versus 264% in  adults following  a single radiolabeled gavage dose of 6.37 rng lead/kg as lead  acetate
(Pounds et al.  1978).  This age difference.may be due, in part, to dietary differences and to the presence
of an undeveloped, selective intestinal barrier to lead in the rat neonate  (EPA 1986a).

Particle size also influences  the degree of gastrointestinal absorption (EPA 1986a; Grobler et al.-1988)
An inverse  relationship was  found between diets containing metallic lead of particle sizes *250 /zm and
absorption in rats (Barltrop  and Meek 1979).  There was a 2.3-fold increase in tissue lead concentration
when animals ingested an acute dose of 37.5 mg/kg with  a particle size of <38 Mm (diameter) compared
 to  a particle diameter of 150-250 /jm (Barltrop  and Meek 1979).  Dermal Exposure

 Inorganic Lead.  Limited information is available regarding absorption after dermal exposure in humans.
 Dermal absorption of inorganic lead compounds is reported to be  much  less significant than absorption
 by inhalation  or oral routes of exposure, because of the greatly reduced dermal absorption rate  (tPA
  1986a).  Following skin application of 203Pb-labeled lead acetate in cosmetic preparations (0.1  mL ot a
 lotion containing 6 mmol  lead acetate/L or 0.1 g of a cream containing 9 mmol lead acetate/kg)  to eight
 male volunteers for 12 hours, absorption was S0.3%, but expected to be 0.06% during normal use of such
 preparations (Moore et al. 1980).   Most of the  absorption took place by 12 hours of exposure.

  Organic Lead. No studies were located regarding dermal absorption of inorganic lead in animals; however
  tetraalkyl lead compounds have been shown to be rapidly and extensively absorbed through the skin ot
  rabbits and rats (Kehoe and Thamann 1931; Laug and Kunze 1948).  A 0.75-mL amount of tetraethyl lead
  which was allowed to spread uniformly over an  area of 25 cm2 on the abdominal skin  of rabbits, resulted
  in 10.6 mg of lead  in  the  carcass at 0.5 hours and 4.41 mg at 6 hours (Kehoe and Thamann  1931).
  Tetraethyl  lead was reported to be absorbed by the skin of rats to a much greater  extent than lead acetate,
  lead oleate, and lead arsenate  (Laug and Kunze 1948).

  2.3.2   Distribution

  Inorganic Lead   Once absorbed, inorganic lead is distributed  in essentially the same manner regardless of
  the  route of  absorption (Kehoe 1987).   This implies that a  common lead transport system is  involved.
  Therefore, the distribution and body burden of absorbed lead for all routes will be discussed in one section.

  Life Sciences, Vol. 46, pp. 569-576
  Printed in the U.S.A.
                                                         PeTgawon Prosa

                    Jaisri Giridhar and Gary E.  Isom

                Department of Pharmacology and Toxicology
                school of Pharmacy and  Pharmacal sciences
                            Purdue university
                        West Lafayette,  IN 47907.

                   (Received in final form December 29, 1989}
                   exposure alters  cardiovascular  'unction  and
         has been  implicated  in  ^he etiology^ of  hypertension.
         Therefore it  was  of  interest  to study  th. short  term
                of  lead  treatment on  atrial  natriuretic  factor
                   hormone which  produces vascular  »"»<»«»  »u«*«

    period water  consumption and urino volume  were
    daily. At  the end of  the 30 day period,  immunoreacfeWe
    levels  of AKF  in hypothalamus,  atria  and plasma  were
    measured  by  radio immunoaesay.  Lead  treatment did  not
    atter  water  consumption,  but  significantly  decreased
    urine output. At all doses,  lead produced a decrease in
    hypothalamic   content  of  ANF  and  slightly  increased
    atrial   levels.  The  content  of   ANF  in  P1""18.  ^|
    decreased.  The changes in ANF content indicate that  lead
    interacts  with  the   hormonal    regulation  «*   JJJ
    cardiovascular system and these  observations may relate
    to the  cardiovascular toxicity of this heavy  metal.

     The observation by  deBold  efc aJU  CD  that  atrial  extracts
produce  potent diuretic  and natriuretic  properties  led  to  the
characterization  of  the  family  of   P^Ptides  known  as  atrial
natriuretic factor  (ANF) .  Subsequently  it has bee" 
             Lead Acetate Interaction with ANF
                                                 Vol. 46, No. 8, 1990
     Related,  exposure  to  heavy  metals  can  induce  significant
changes  in cardiovascular  function.  Lead has  been  implicated in
the pathogenesis  of hypertension following both acute and chronic
exposure  in  animals and humans  (7-11). Additionally, hypertension
has  a  high  incidence  among  workers  exposed  to  lead,  possibly
related  to  lead-induced renal  impairment  (10).  Several studieo
have   associated   a   direct  relationship   between  blood   lead
concentration and blood pressure (7,  11).  Also  elevated blood  lead
levels  have  been  related  to  an  increased  risk  of  myocardial
infarction (12).  Elevated ronin-angiotensin activity was detected
following long tarm  exposure  to lead  in  dogo  (13)  and acute and
chronic exposure in  rats  (14,  15) .  Acute lead administration can
produce a transient decrease in plasma renin activity followed by
a  prolonged  elevated  activity as  a  result  of  increased renin
secretion (14) .  Chronic exposure to  lead increased basal  plasma
renin  activity and renal ranin concentration  (16). Also in  some
high renin hypertensive patients,  high blood lead levels  have  been
detected  (16). It  has  been proposed  that the mechanism of lead-
induced hypertension may involve the inhibition ol vascular sodium
and   potassium   pump,    where  impaired   sodium   and   potassium
cotransport produces increased intracellular sodium content, which
as result of sodium-calcium exchange  results  in  increased  calcium
levels in vascular tissue  (17,  IB).  Since ANF plays an  important
role  in blood pressure regulation,  the present study addressed the
effects of lead  on immunoreactive  levels of ANF  in plasma, heart
and bypothalamus.


Animals;  Twenty  male  Sprague  bawley rats  (Harlan, Indianapolis,
IN.)  .(IS0"175 g  in weight) approximately  5 weeks old were randomly
divided  into  5 groups  of 4 animals each  and  housed in metabolism
cages (9  by  10.5 by  9  inches).  Tap water and laboratory chow  were
made  available  to  tho  animals  ad   1 ibituro   .  The animalr.  were
 injected  intraperitoneally with  different  doses  of lead  (0.01,
 0.1,  0.5  and 1.0 rag/Kg of body  weight) daily for 7  days at 900 and
 2100 hrs.  Control animals  received normal  saline.  Following  lead
 treatment tho animals were maintained for an  additional  30  days
 during  which water  consumption and  urine volume  wer«i monitored

 preparation  of plasma  extracts: At the end of  the treatment period
 the animals  were sacrificed and the  trunk blood was collected  in
 polystyrene  tubes  containing  0.5 ml of a  solution containing  0.47*
 sodium   chloride,    1%   ethylenediamine   tetraacetic  acid,  0.1%
 bacitracin,   30   iM   phenylmethylsulphonyl fluoride   and   1   pK
 aprotinin. Blood was immediately centrifuged at 1500 g at  4°C for
 15 min.  Plasma was separated  and ANF  was  extracted, following haat
 inactivation of  interfering plasma proteins,  as  reported by linuma
 et al.   (19).  Briefly, 0.5 ml  o.f  plasma was  mixed with an  equal
 volume   of  0.1   M  acetic  acid  solution   containing   protease
 inhibitors.  The mixture  was  heated  at  85°C for  10  min and then
 centrifuged  at  800 g for 10 min.  The  supernatant was separated and
 stored  at  -70°C.  Aliquots  of 100  pi   of the  extracted plasma
 supernatant  were used  for radioimmunoaseay.

 Preparation  of  atriaj  extracts;  Atrial extracts  were  prepared
 according  to  the  method   of  Gutkowska  et  al.  (20) .   After
 dec.- j
 at :j
 at -j
 for !


Vol. 46, No. tf,  1990
                           Lead Acetate Interaction with ANF

for ANF by RIA.

                Izfo?0 g  forY30 »in. suitably  diluted and aosaycd
                                             The  hypothalami   were
 atria   and*    T hpothaa3nc extracts, ware  roade  and i  »«J
 ralioimmunoassay. Protein determinations -wore  mada by the modified
 method of Lowry  (22) .
WJ.«. rat  ANF(1-28).  'in  order to prevent  interference  by lead,
EDTA (1 mM) was added to  the samples.

g«-.«fcisti«»i.' analysis: The data were subjected to one-way ANOVA and
then analyzed by  Duncan's multiple ranges test.


     At the end  of  the treatment period  lead treated animals did
     dilpla? observable signs of toxicity.  Plasma ANF levels were
     ea-jed in  all  lead treated animals as compared to control value
To^le  +  0.037  ng/«H   of  plasma)-.  Except  for  the  001  mg/kg
treatment group,  lead  produced a dose  related decrease  in plasma
ANF  Statistically  the  1.0  mg/kg dose of  lead was significantly
decreased  (P < Q.05) as  compared to the control value (Fig. 1).





                            0.01     0.1

                                 FIG.  1

        Effect  of  varying  doses  of  lead  on  ir-ANF levels  in.
        plasma,  expressed  as  percent control ±  SEM  (ng/ml).
        Each  value is  the  mean  of 4  animals.   *significantly
        different  from  control  (P  < 0.05).

                                                            n u. y
                                                                                       U X  I  . W —'
                             Lead Acetace Interaction with ANF
                                                                  Vol.  46. No. 8, 1990
                     Atrial ANF level at  0.5 ing/kg dose  was significantly  higher
                (p <  0.01) than that  of the  control  (SB. 359  i  23.399 fig/gro  of
                atrial tissue). The  other doses were  not significantly  different
                from the control value (Fig. 2).

                     In the hypothalaaus  lead  produced a dose related decrease  in
                ANF content as  compared to control values  (703.352  ± 203.937 ng/gm
                of hypothalaroic tissue)  and this  decrease  for  the 0.01, 0,5  and
                1.0 mg/kg  was significantly  different (P <  0.05) from the  control
                value  (Fig. 3)-





                          Fig. 2

Effect  of varying doses  of lead  on ir-ANF  content  in
atria,  expressed  as  percent  control  ±  SEM  (Ig/gro  of
atrial  tissue) .   Each value  is the mean  of  A  aniTnale.
**Significantly different from  control  (P  < 0.01).





                                                  DOSE (mg/kg)

                                                Fig. 3

                      Effect  of  varying  doses  of  lead, on  ir-ANF content  in
                      hypothalamus,. expressed as  percent control + SEM (ng/gm
                      of  hypothalamic  tissue) .    Each  value is  the mean  of 4
                      animals.    *Signifieantly  different  from  control   (P <
                                                                                             Vc i
                                                                       th !
                                                                       wa j
                                                                       la j

 EX) j


 LEV |


                           C.L. • J. — <4 0.
                                          ~ i <4OO
                                                          i-iug  uo . a f
                                                                                INO . UUi
                                                                                           . UD
 Vol.  46. No. 8, 1990
                            Lead Acetate Interaction with ANF
      No  change  in  the  water  intake  was  observed  between  the
 control  animals and the  lead treated  animals at all dose  levels
 throughout the  experimental period (Table 1).  However urine output
Evas significantly  decreased in all the lead treated animals on the
 •last  treatment  day (day  7)   and  at  the end of the  maintenance
         (Table 2).

                                TABLE I

 Twenty-four  Hour wator Consumption of Control and Lead Treatment
* Exp Group

..-1S»D (0

. 0






. 48




3 .





4 .13
  Water consumption of control and  lead  treated groups,  expressed as
  ml •+• SEM (total consumption over  24  hrs) ,  on day 0 (day before the
  first lead treatment),, day 7 [last day of  treatment with either
  saline (control) or load  (treatment  groups)] and day 37 (last
  experimental day).  Each value  is the  mean of 4 animals.

                               TABLE II

  Twenty—Tour Hour Urine Volume of  Control and Lead Treatment Groups

7 .

-g) 5,
r) 7.
r) 6.

!) 6.



(0) :














. 33

(7) Dav f37J.

± 0.

± 0-
± 1.
± o.

± 1.







00 ±

50 £.
25 ±
38 ±

25 +






  Exp. Group


  LEAD '(O-01 m

  LEAD (0.1 wg/kg)

  LEAD (0.5 mg/kg)

  LEAD (1.0 mg/kg)
  Urine volumes  of control and lead treated groups, expressed as ml
  ± SEM  (total urine volume over 24 hrs),  on day 0  (day before the
  first treatment),  day 7 [last day of treatment with cither saline
  (control)  or ]ead (treatment groups)] and day 37  (last
  experimental day).  Each value is the mean of 4 animals,
   "significantly different from control (P < 0.01) on respective

                             Lead Acetate Interaction with ANF
                                                                 Vol.  46, Mo. 8,  1990
                                             TABLE III

                Body Weight Measurements  of  Control  and  Lead  Treatment Groups







Pav f 7 > Dav


5 ±
5 ±
5 ±
0 ±
5 ±



327 .



Body weights  of control and lead treated groups, expressed as
grams ±  SEM,  on day o (day before the first treatment) ,  day 7
[last day  of  treatment with either salino (control)  or lead
(treatment groups)] and day 37 (last experimental day).   Each
value is the  mean of 4 animals.   Significantly different from
control  (P <  0.05)  and    significantly different from control (P
<  0.01)  on respective days.


     The present study was designed  to  investigate  the  short ten
effect of  lead on  cardiovascular  system and subsequent  changes in
tho ANF  system (23).   Lead produced significant alterations in ANF
immune-reactivity  with  dose-related  decreases  observed  in  the
plasma  and hypothalamus  and an  increase in atria.  These changes
may result from the pathological effects of lead, since  changes in
ANF levels have been  reported in hypertension  and  kidney disease-,
(6, 24).

     As  the. Riecha-: sm  of  lead-induced toxicity  is  not  completely
known, it  is  not clear as to how lead acts on the ANF system. Lead
may  act  at  one  or  more  sites  involved  in   regulation  of  the
hormone. Lead may have direct actions on ANF synthesis and release
both  in  hypothalamus  and  atria.  Additionally,  it - may  indirectly
modify   ANF  levels   in   hypothalamus  and  atria,  by   altering J-
heurotransmitter systems such as norepinephrine and  dopamine (25),'.-'"'
which are  known to modulate  ANF levels  (26). Also  changes  in ASF^-
levels in  atria and plasma may be brought  about  by morphological^
and biochemical alterations of the heart,  and  changes  in vascular
smooth muscle reactivity caused by lead (17-18,  27-29).            g>

     Lead-induced nephrotoxicity  has been  well  characterized  in^f
animals  and humans (7, 30). Lead  produces  proximal  tubular damage^'
and epithelial cellular necrosis  (30) .  These effects may  lead to''
changes  in sodium transport and nance resulting in  changes  in AN
content  through impairment of kidney  function and  altered  flaid
balance.   In  the present study indicies of nephrotoxicity were not
determined.   However,  the results suggest impaired renal function.
                                                                                          VoJ. •
                                                                                          dec; •
                                                                                          bodj !
                                                                                          tre; |
                                                                                         •Btr.i i
                                                                                          levc :











           i j. i_j i -t  i\ t
                                                           nuy uo > 3i
                                                                                  nu . uu a.  r .
•Vol.  46, No. 8, 1990
                           Lead Acetate  Interaction with ANF
     There was no change  in  the water intake in the lead treatment
groups as  compared  to controls at all dose  levels  throughout the
experimental  period*   This  was  accompanied   by  a  significant
decrease  in the  urine  output.   Decreased 'urine  output  without
accompanying  decrease  in  water  consumption   leads   to  fluid
retention  in  lead treated animals.   Also  significant increase in
body weights  of  lead treated  animals is  seen on  day  7  (end of
treatment  period)   and on  day  37  (end  of  experimental period),
which may  be explained  by  fluid  retention.   This effect  may be
related to Jead-induced  nephrotoxicity.  Expansion of fluid volume
can produce  changes  in  ANF and may  be a  contributing  factor for
altering the  ANF system. ANF  levels in related cases of impaired
kidney function  are generally  elevated in  plasma  (2$).  However in
this  particular  case ANF  levels  in  plasma are  lower than  the
control value.  Hence  lead may decrease the release of ANF  from
atria and  this  would account for  the observed elevated atrial ANF

     In  conclusion  the  present study indicates  acute treatment
with  lead  acetate  can produce significant  alterations in select
tissue levels of  ANF.  The significance oC this action of the heavy
metal is presently  being investigated.


  S .



 11 .






Life Sciences 20.  89-94 (1981).
S.W. HOMUERG & P.  NKEDLEMAN,  Science  221, 71-73 (1983).
A.J. osBOLD, Science 230.  767-770 (1985).
Biophys. Res. Commun.  127.  413-419  (1985).
S.  SUIONO,  K.   NAKAO,  N.   MOR11,  T.   YAMADA,  H.   ITOH,  M.
Biochem. Biophys.  Res.  Commun.  13S.  728-734  (19SG).
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Coromun. UL1, 759-765  (1985).
Amer. J. Epidemiol.  17.3.  8OO-808 (1986).
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J,  Lab. Clin. Med.  99.  354-362  (1982).
Eng. J. Med. 309,  17-21 (1983).
L.C. 11ARLAN, J. Amer.  Med.  Assoc. 2-^3.t   530-534 (1985).
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B.P.  FINE, T. VETRANO, J.  SKURNICK  &   A. TY,  Toxicol . Appl.
Pharmacol. 93, 388-393 (1988).
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Pharmacol. 46, 435-447 (1978).
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759-77O,  (1980).
A.J.VANDER, Environ. -Health Perspec.. 7_8, 77-83 (1988).

nuun i mix K.C.O i I K.  i ELL • i ~*4 iu~ J3u~ m Jo
                                    u u q  u o_
                                                                             INU . u u i r . u 3












             ' 30.
       Lead Acetate Interaction with AUK
                                             Vol. 46, No. 8, 1990
E.  WEILER,  F.  KHALIL-MANESH  &  H.  GONICK,  Environ.  Health
Perspec. 76.  113-115  (1988).
CLAUDE,  B. JUGUET, B.  FESTY &  J.  LELLOUCH,  Environ.  Health
Parspec. 78,  47-51 (1988),
& Y. KOMAHARA,  Clin.  Chem.  12,  674-676  (1987) .
GENEST, Biochem.  Biophys. Res.  Commun.  121,  593-601  (1984).
J.  GLOWINSKY   &   L.L.  IVERSEN,  J.   Neurochem.  H,  655-659
{ X966) .
G.L. .PETERSON',  Analy.  Biochem.  8_3_, 346-356 (1977)
357-364  (1986).                                          **'
Lab. Invest.  48.  347-355,  (1988).
M.K. SHELLENBERGER, Neurotoxicoloqy,  5  177-212  (1984)
1545-1551  (1987) .   ,.                                      '
             '•J- WINQU1ST,  W.  VICTERY  &  A.J.
9i-99K°1988?"T"  BARR°N  & J-p'  TOM-  Environ.  Health Porsp.  2ft,
D.P. BERNARD  & C.E. BECKER,  Clin. Toxicol . 2£,  3-34 (1988)..

                                                      L»l|g3T ^^J^^Ji^^^'Vv^'V^^^^/--,-^

                                                                                                 k!" 5r- -*•: itr".

                                                            ^^«^T>.-S^M* .-^-'^-^fa^^^a"*' ^Hr^^'^'m.^^'.f-^^- ^^^.^ga*gg»gT5aeag»:l.-g-.--^'..
                                      ; t.«..-^
CL1N. CHEM. 32/7, 1255-1363 (1986)
The Porphyrias: Recent Advances
J. Thomas Hindmarsh
                  NOTICE: This Material May
                   Be Protected B-, Copyight
                    Law {Title 17 US. CcJa)
Recent research has elucidated several of the hitherto poorly
understood steps in heme synthesis. This review describes
this metabolic pathway and pinpoints the enzymatic block-
ages  in  the  various  porphyrias. Recent advances in the
understanding of the etiology of porphyria cutanea tarda are
discussed, as are the abnormalities of porphyrin metabolism
seen in chronic renal failure and in lead poisoning. An outline
is given of the clinical and biochemical abnormalities seen in
the porphyrias.  Included is an algorithm to aid in {he differen-
tial diagnosis of these diseases, and a brief review of the new
analytical techniques  available  for  the identification and
quantification of porphyrins  and their precursors in  body

Additional Key phrases: porphyria cutanea tarda  •  erythmpoietic
porphyria  •  lead poisoning •  chronic renal failure  • metabo-
lic pathways  •  heme synthesis •  neiirotoxicity  • hepatic iron
accumulation  •  alcoholism  •  estrogen-induced effects •  her-
itable disorders  •  harderoporphyria

Heme Synthesis
  The principal sites for heme synthesis in the human are
the hcjnapoietic tissues and the liver. The synthetic path-
way is a series of irreversible reactions, some of which occur
in the cell mitochondria and some in the cytoplasm (Figure
1). Intramitochondrially, the'reactions are mainly oxida-
tion—reduction, whereas the extramitochondrial  steps are
condensation and decarboxylation. The enzyme 5-aminole-
vulinate (ALA) synthase (EC exhibits  much less
activity than the other synthetic enzymes and controls flow
through the pathway; its activity is inhibited by heme or
hemin. After release from the enzyme surface, S-aminolevu-
linic acid diffuses into the cytoplasm and, catalyzed by ALA
dehydratase (EC, two molecules condense to form
the mono-pyrrole porphobilinogen. ALA dehydratase is a
zinc-dependent metalloenzyme, and zinc partly protects this
enzyme against the adverse effects of lead in  vitro CO and
possibly also in vivo (2).
  The enzymes  porphobilinogen  (PEG)  deaminase (EC and uroporphyrinogen-in synthase (EC act
together to polymerize four molecules of porphobilinogen to
form  the cyclic tetrapyrrole, uropbrphyrinogen IE.  PEG
deaminase appears to catalyze the  condensation of four
PEG molecules in a symmetrical head-to-tail arrangement
to form the tetrapyrrole, hydroxymethylbilane (Figure 2).
  Division of Biochemistry, Ottawa Civic Hospital, 1053 Carling
Ave., Ottawa,  Ontario, Canada K1Y 4E9; and Departments of
Biochemistry and Pathology, University of Ottawa.
  Received February 25, 1986; accepted April 7,1986.

             ALA Synth*** •«


   1      I    ALA Dehydratase



                                            Negative Feedback

       UropOfpbyrmogen 4


       Coproporphynnogen 5
           Oxtdase    „

       Coproporpnynnogen 6


       ProtopOTphyrinogen  7

        FcfroehelaUce  8

        F«~ — HEME	
               UROPORPHYRINOGEN I,

 Fig. 1. The pathway for heme synthesis, including sites of enzyme
 insufficiency in the porphyrias
 J, ALA dehydratase deficiency, tyrostnemia, toad poisoning; 2, acute intermittent
 porphyria; 3, .congenital etythropoietic porphyria; 4, poiphyria cutanea tarda,
 hepatoerythropoietic porphyria, toxic porphyria; 5, hereditary ccproporphyria, toad
 poisoning; 6, harderoporphyria; 7. porphyria variegata; 8, protoporphyria, tead
 poisoning. * = probable step
 The enzyme uropofphyrinogen-in synthase then in some
 way "flips" the D pyrrole to an asymmetrical arrangement
 and closes the porphyrin ring (3, 4). In the  absence of
 uroporphyrinogen-in synthase, hydroxymethylbilane spon-
 taneously cyclizes to form the I isomer of Uroporphyrinogen.
 Support for this synthetic pattern comes from the observa-
• tions that uroporphyrinogen-in synthase cannot consume
 PEG as substrate nor can it convert the I isomer of Uropor-
 phyrinogen to the HI isomer. Ordinarily, excess uroporphyr-
 inogen-in synthase is  present, which greatly favors the
 formation of the ffl isomer series. Its activity in human and
                                                                          CLINICAL CHEMISTRY, Vol. 32, No. 7, 1986  1255

                       UROPORPHYRINOGEN HI
Fig. 2. Formation of uroporphyrinogen from porphobilinogen
A acstata: P, prcpionata
pig livers reportedly is inhibited in vitro by ferrous com-
pounds (5).
  The decarboxylation of the acetate side-chains of uropor-
phyrinogen DI appears to be a sequential process proceeding
through 7-, &-, and 5-carboxyl intermediates; it is catalyzed
by the  enzyme uroporphyrinogen  decarboxylase (EC The acetate side-chains of uroporphyrinogen I are
also decarboxylated, ultimately to form coproporphyrinogen
I, after which the metabolic pathway of this isomer series
proceeds no further. Uroporphyrinogen decarboxylase can
react only with uit>]5orphyrinogen as substrate and not the
corresponding porphyrin. Ferrous compounds are reported
to inhibit the enzyme in vitro (6, 7), although one study has
reported the opposite (8).
   Coproporphyrinogen oxidase (EC converts copro-
 porphyrinogen HI to protoporphyrinogen, in which the pro-
 pionate side-chains in the A and B pyrrole rings are oxidized
 and decarboxylated to vinyl  groups. This reaction probably
 proceeds with the preliminary formation of harderoporphyr-
 inogen, an intermediate  compound  with only one vinyl
 group (on the A pyrrole). Protoporphyrinogen is oxidized to
 protoporphyrin by protoporphyrinogen oxidase (EC
 The final step  is chelation of protoporphyrin  with ferrous
 iron to form heme, catalyzed by the enzyme ferrochelatase
 (EC Other divalent metals (cobalt,  zinc, copper)
 also may be chelated with protoporphyrin.
   Control of the rate of heme synthesis is achieved largely
 by a negative-feedback effect of heme on the enzyme ALA
 synthase. PEG deaminase also has a lower activity than the
 other enzymes in the pathway and probably also  exerts
 some control. ALA synthase activity appears to be increased
 as a compensatory effect in most, possibly all, of the porphyr-
 ias, and this action is responsible for the excess production of
intermediary metabolites proximal to the enzymatic blocks'
in these diseases. Brodie et al.  (9) and Moore (10)  have
postulated that variability in the activity of PEG deaminase-
explains the different clinical features of the acute and
nonacute porphyries.  Their thesis is  that in the  acute
porphyries, ALA and PEG accumulate in the tissues, where-
as this is not a feature of the nonacute porphyrias; moreover,
this accumulation in nervous tissue is probably responsible    }
for the neurotoxic features of the acute disease. Because
heme synthesis is potentially suboptimal in all porphyrias,
these diseases carry the potential for an accumulation of
excess intermediary metabolites  proximal to the enzymatic
block. However, in the nonacute porphyrias,  PBG deami-  -
nase activity appears  to be increased (at least hi erythro-
cytes), thereby preventing the accumulation  of porphyrin.  /
precursors (ALA and PBG), whereas in the acute porphyrias  •
the activity of this enzyme is normal (hereditary  copropor-
phyria and porphyria variegata) or subnormal (acute inter-
mittent porphyria), and  therefore excess ALA and PBG
accumulate. Although this is an attractive hypothesis, it is
not certain that heptatic PBG deaminase is  similarly in- •
creased in the nonacute porphyrias, and further work is
needed before the theory can be fully accepted.
   The mechanisms whereby the porphyrin precursors (ALA
and PBG) produce neurotoxicity have not been fully eluci-
dated, but clinical neuropathy is related to their accumula-
tion. Neuropathy occurs in diseases hi which ALA alone   .
 accumulates (ALA dehydratase deficiency); ALA or a me-
tabolite may be the  responsible: agent. There have been
problems in producing comparable neurotoxic effects in vivo
 in experimental animal models (11), but toxicuy may be
 related to the ability of ALA to inhibit competitively the
 binding of the central nervous system neurotransmitter, y-
 aminobutyric acid,  to synaptic membranes in brain tissue
 (12,13).                                   .         .
    Factors known to precipitate acute attacks in porphync
 subjects include alcohol, stress, infection, starvation, hor-
 monal changes, and the administration of certain drugs. It is
 not  completely understood how drugs  precipitate acute
 attacks. The mechanisms may be related to the depletion of
 the mitochondria! free-heme pool by decreased heme syn-
 thesis or increased  utilization of hemoproteins such as
 cytochrome P450, resulting in activation of cellular ALA
 synthase (11).

 The Porphyrias

    The porphyrias  traditionally are classified according to
  whether the liver  or the erythropoietic tissue is the main
  source of excess porphyrin production  (14), and this ap-
  proach has some merit it is simple. The classification shown
  in Table 1 is based on whether patients with the diseases
  suffer acute attacks, dermatological manifestations, or both.
  Any classification should include an account of the related
  enzyme deficiencies.

  Congenital Erythropoietic Porphyria
    Patients with this rare disease suffer from mutilating
  photo-induced  skin lesions, are excessively hirsute, and
  often have hemolytic anemia.  Fluorocytes—erythrocytes
  exhibiting fluorescence when activated with the  appropriate
 ' light energy—are present in the blood, and the teeth and
  bone  marrow  may also  exhibit fluorescence'. Studies of,
   erythrocytes and skin fibroblasts have shown the activity of
   uroporphyrinogen-in synthase to be markedly decreased in
  1256 CLINICAL CHEMISTRY, Vol. 32, No. 7, 1986

                                     Table 1. Classification 'of the Porphyrias
Nonacute porphyrias:
Porphyrias producing cutaneous lesions
Congenital erylhropoietic
Porphyria cutanea tarda

Toxic porphyria

                                    Mode of Inheritance
Autosomal recessive

Sporadic and autosomal

Autosomal recessive

Autosomal recessive
Autosomal dominant
Acute porphyrias:
Porphyrias producing neurological symptoms
Acute intermittent porphyria          Autosoma! dominant
ALA dehydratase deficiency          Autosomal recessive

Porphyrias producing both neurological and cutaneous manifestations
Variegate porphyria                 Autosomal dominant
Hereditary coproporphyria           Autosomal dominant
                                    Enzyme defect
  decarboxyiase'  -
Coproporphyrinogen oxidase
                              Porphobilinogen deaminase
                              ALA dehydratase
                                (porphobilinogen synthase)

                              Protoporphyrinogen oxidase
                              Coproporphyrinogen oxidase
                                                               Predominant sitefs) of
                                                               metabolic expression
Erythroid cells



Erythroid cells and
Erythroid cells and
this condition, resulting in overproduction of the I isomers of
Uroporphyrinogen and Coproporphyrinogen. Excessive uro-
porphyrin and coproporpnyrin (the I isomers) are found in
the urine.
   Pimstone and Kushner (15-17) described an unusual case
of congenital erythropoietic porphyria in which erythrocyte
uroporphyrinogen-in synthase activity was within normal
limits but  Uroporphyrinogen decarboxyiase  activity was
subnormal by approximately 50%. Urinary porphyrins were
mainly the HI  isomers, and fecal  isocoproporphyrin was
greatly increased. Seventy percent of bone marrow normo-
blasts showed nuclear porphyrin fluorescence; liver porphy-
rins were  not increased. These authors believe  that two
inherited abnormalities coexist in this patient:  erythropoi-
etic Uroporphyrinogen decarboxyiase deficiency and a dys-
erythropoietic anemia.

Porphyria Cutanea tarda- (PCT)
   This disease is associated with reduced liver Uroporphyr-
inogen decarboxyiase activity. There appear to be two types:
a sporadic, and a rare inherited form (familial PCT). In the
latter, erythrocyte uroporphyrinogen decarboxyiase activity
 is also reduced.(IS, 19). The enzymatic defect in familial
 PCT is inherited as an autosomal dominant trait (20). One
 study of familial PCT has  reported  that the amount of
 immunologically  reactive enzyme  in erythrocytes is de-
 creased along with the enzymatic activity (21).
   Sporadic PCT  is  the  commonest  porphyria  in North
 America. Its onset is often precipitated by ethanol overin-
 dulgence or, more rarely, by therapy with estrogen or use of
 the contraceptive pill. However, only  a small proportion of
 persons exposed to these substances is affected, indicating
 that other etiological factors are also  operating.
•  The role of iron in the causation of PCT is  confusing and
 controversial. Most patients with active PCT have increased
 hepatic iron stores;  hepatic siderosis was present in 80% of
 the patients  in the study  of  Grossman et al.  (22).  The
 increase in hepatic iron varies  from a  modest twofold
 increase to concentrations similar to those seen in idiopathic
 hemochromatosis and appears in many cases  to be the
 consequence of increased intestinal absorption (23). The iron
                          is especially prominent in the hepatic parenchymal cells and
                          the portal tract macrophages, less so in the Kupfier's cells
                          (23). Decreasing hepatic iron stores by repeated phlebotomy
                          produces clinical and biochemical remission of the disease—
                          amelioration of the symptoms and signs and a decrease in
                          urinary uroporphyrin excretion (24). Also, Sweeney et al.
                          (25) have shown that mice rendered iron-deficient by repeat-
                          ed  bleedings  are protected against the porphyrinogenic
                          effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin. However, iron
                          plays a "permissive" rather than simply a causal role in
                          PCT, because even though alcoholics  commonly have  in-
                          creased hepatic iron stores (26), the etiology of which is
                          unclear, this finding is more common  than PCT is in this
                          group. PCT is also uncommon in patients with idiopathic
                          hemochromatosis. Another finding irreconcilable with a
                          simple  causal role for iron is that, although phlebotomy
                          produces clinical improvement in patients with PCT, it does
                          not increase the  low activity of hepatic uroporphyrinogen
                          decarboxyiase in these patients (27). Hepatic siderosis does
                          not appear to be limited to the ethanol-induced variety of
                          PCT, because it was reported in at least one patient with
                          estrogen-associated PCT (28). Thus iron overload cannot be
                          attributed solely to  alcoholism.  How often hepatic  iron
                          stores are completely normal in active  PCT is not clear, but
                          it probably is uncommon. Hepatic iron accumulation was
                          not seen in the study of Elder et al. (29)  of rats rendered
                          porphyric by hexachlorobenzene, so increased iron loads are
                          not essential for porphyria development,  at least in their
                          model. The  protective effect of phlebotomy in a similar
                          mouse model has already been mentioned (25).
                             The  etiology  of PCT  has  recently been reviewed by
                           Pimstone (30) and Sweeney (31); clearly, it is multifactorial.
                           The key problem is failure  to decarboxylate uroporphyrino-
                           gen adequately. Uroporphyrinogen decarboxyiase activities
                           are decreased by about 50%  in livers of patients with
                           sporadic PCT and in the livers and erythrocytes in familial
                             In familial PCT there is a  clear pattern of autosomal
                           dominant inheritance of the enzymatic defect, and Sassa et
                           al. (32) have shown that the immunoreactive enzyme con-
                           tent of erythrocytes, at least, is  similarly decreased. This
                                                                          CLINICAL CHEMISTRY, Vol. 32, No. 7, 1986  1257

disease, then, is associated with an inherited deficiency of
uroporphyrinogen decarboxylase  activity  and synthesis.
However, the disease is not clinically expressed in all family
members who inherit the enzyme deficiency, so other factors
such as increased hepatic iron stores and alcoholism must
contribute to the impairment of uroporphyrinogen decarbox-
ylation.                                        .  .    .
  The etiology of sporadic PCT is more complex. Diminished
hepatic uroporphyrinogen decarboxylase activity does not
recover, even though the clinical state improves when iron
stores are reduced by phlebotomy, so these patients also
seem to have an irremedial enzyme defect. This may also be
inherited, but the pattern of inheritance is not clear. As was
the case in familial PCT, the enzyme deficiency in sporadic
PCT  does not produce clinical disease unless the patients
also have hepatic siderosis and (or) have some other disease-
precipitating factor such as alcoholism or take estrogens.
  An interesting hypothesis, supported by HLA marker
studies, has been advanced by Kushner et al. (33). They
propose that, in sporadic PCT, a defect in uroporphyrinogen
decarboxylase is inherited as a recessive trait. To develop
the disease, the patient must be homozygous for the enzyme
defect and also inherit one allele, perhaps on a different
chromosome—i.e., be  heterozygous—for the autospmal re-
cessive disease, idiopathic hemochromatosis. Hepatic sidero-
sis,  perhaps the consequence of the recessive state  for
hemochromatosis, has a further inhibitory effect upon the
already compromising  enzyme  (33, 34>.  It  is  not  clear
whether alcohol and estrogens aggravate the enzyme insuf-
ficiency by further inhibiting the enzyme directly or by
promoting further hepatic iron deposition. All these factors
 combine to produce clinical disease from a relatively innocu-
 ous  enzyme deficiency. The complex  inheritance pattern
 explains  why  sporadic PCT does  not appear in several
 members of a family; the autosomal dominant inheritance of
 the enzyme deficiency in familial PCT explains why it does.
   The complex interactions of the etiological factors in PCT
 have also recently been reviewed by Mukerji et al. (34).
 They suggest  that iron may further  compromise uropor-
 phyrinogen decarboxylation, in  two ways. Firstly, ferrous
 iron directly inhibits uroporphyrinogen decarboxylase by
 combining with sulfhydryl groups on the enzyme. Secondly,
 in the presence of oxygen, binding of ferrous iron to cellular
 sulfhydryl groups promotes the generation of superoxide
 radical anions, which might further inhibit enzyme activity
 and (or) oxidize porphyrinogens to porphyrins that cannot be
 acted upon by the enzyme.
   The symptomatology of PCT is  very  variable but the
 patient usually shows some cutaneous blistering and fragil-
 ity in the light-exposed areas, along with some hirsutism.
 Diagnosis is usually made from a combination of a classical
 clinical picture and increased urinary  porphyrins, with
 uroporphyrin  exceeding coproporphyrin.  It is  sometimes
 difficult to distinguish patients with PCT from those with
 porphyria variegata, as there may be no clear history of
 inheritance in the latter disease. In porphyria variegata the
  urinary excretion of coproporphyrin usually exceeds that of
  uroporphyrin, but in the phase of recovery from an acute
  attack, urine uroporphyrin may exceed coproporphyrin,
  perhaps because of nonenzymatic conversion of porphyrin
  precursors to uroporphyrin. If there is no clear history of
  ethanol abuse, I usually use thin-layer chromatography to
  look for the presence of grossly increased amounts of isoco-
  proporphyrin in the feces of patients suspected of having
  PCT (35).
  Isocoproporphyrin is formed in excess in PCT, probably by
the following mechanism (36). Uroporphyrinogen decarbox- _
ylation occurs in a step-wise manner,  starting with the
acetate group of the D pyrrole (Figure 3). Because flow
through this pathway is impeded, owing to impaired uropor-
phyrinogen decarboxylase activity, 7-, 6-, and 5-carboxylic
porphyrins accumulate. Heptacarboxylic porphyrin appears
in excess in the urine  and can be used as a biochemical
marker for PCT. Isocoproporphyrin is a porphyrin with four
carboxylic groups in which one propionate group has under-
gone oxidative decarboxylation before the last acetate side-
chain has been decarboxylated. Its formation in excess in
PCT is probably the consequence of the action of copropor-
phyrinogen oxidase on the excess pentacarboxylic porphyr-
incgen. This enzyme converts the propionate group of the A
pyrrole of pentacarboxylic porphyrinogen in the liver to a.  ,
vinyl group. The resulting compound (dehydroisocopropor-
phyrinogen) is then excreted  into the feces, where fecal
bacteria reduce the  vinyl group to an ethyl group. This
pathway is insignificant in normal healthy persons (37).
   A PCT-like syndrome is occasionally seen in chronic renal
disease, particularly in patients who are undergoing regular
hemodialysis (38). In some cases, results of porphyrin stud-
ies  have been found to be within normal limits, and the
disease has been called "pseudoporphyria"; some cases of
pseudoporphyria have baen attributed to use of the drugs
nalodixic acid, tetracycline,  and furosemide. Other studies
 have reported distinct abnormalities in porphyrin metabo-
 lism, including increased porphyrins 'in plasma and urine
 and diminished activity of uroporphyrinogen decarboxylase
  Fig. 3. Formation of coproporphyrinogen and isocoproporphyrin from
  —<-, normal pathway; —>, pathway in porphyria cutanea tarda. A, acetate;-P,
  praptonate: V. vinyl;  M, methyl. Compounds in  which  R  =  hydroxyettiyt
  (CH3CHOH) and desethyi (H) are also formed
  1258  CLINICAL CHEMISTRY, Vol. 32, No. 7, 1986

in erythrocytes. Interpretation of the biochemical indicators
of porphyrin metabolism is difficult in renal failure, when
urinaiy excretion of porphyrins may be impaired and por-
phyrin concentrations in plasma correspondingly increased.
Day et al. (39,40) have recently reviewed porphyrin metab-
olism in chronic renal disease. Their studies show that
uroporphyrin concentrations in plasma and urine are com-
monly supranormal in chronic renal failure, whereas values
for  coproporphyrin in  urine and plasma are often subnor-
mal. Plasma uroporphyrin concentrations in  two of their
three patients with renal failure and bullous skin lesions
were similar to those reported for patients with PCT from
other causes. However, the plasma uroporphyrin concentra-
tion in the  third patient with skin  lesions, although in-
creased, was similar to those seen in some dialysis subjects
without skin lesions. Poh-Fitzpatrick et al. (41) have also
reported overlap of plasma uroporphyrin concentrations in
patients with  chronic renal failure, with or without skin
  Day and Bales (39) reported that the increased uropor-
phyrin in chronic renal disease was the EH isomer, in
contrast to sporadic and familial PCT, in which the I isomer
is usually found in excess. Uroporphyrinogen did not pass
throrgh the dialysis  membrane, probably because of its
binding to a plasma protein. Another surprising finding in
their study (39) was the markedly decreased coproporphyrin
concentrations in urine and plasma, which, associated with
decreased fecal porphyrins, led them to conclude that the
kidney might be  a major  source of coproporphyrin. In
support of this thesis, they reported a patient with porphyria
variegata who underwent renal transplantation .for renal
failure (40). The patient showed no accumulation of plasma
coproporphyrin before transplantation, despite cessation of
urinary copniporphyrin'excretion; marked coproporphyrin-
uria occurred after transplantation.  Why a kidney from a
presumably nonpoFphyric donor would produce  excess co-
proporphyrin  when transplanted into a pt^.hyric patient
 remains to be explained, as do many of the other features of
 this interesting hypothesis. Etiological factors contributing
 to  the increased prevalence-of PCT in chronic renal disease
 might include the  iron overload sometimes seen in these
 patients as  a consequence of therapy with iron and repeated
 blood transfusions (42). Aluminum is known to inhibit some
 enzymes in the heme synthetic pathway and, because it
 appears to be retained in some patients with chronic renal
 failure receiving aluminum hydroxide therapy, it may play
 some part as  an etiological factor (43).

 Toxic Porphyria
    This disease resembles PCT. Hepatic uroporphyrinogen
 decarboxylase concentrations decrease in response to expo-
 sure to a toxin such as hexachlorobenzene or 2,3,7,8-tetra-
 chiorodibenzo-p-dioxin. However, the prevalence of porphyr-
 ia in persons exposed to these toxins is much higher than in
 ethanol-.and estrogen-associated PCT, and the mechanism
 presumably is a more direct effect of the toxin on the
 enzyme. Elder and Sheppard (44) have shown, in a rat-liver
 model, that the immunoreactive uroporphyrinogen decar-
  boxylase content remained unchanged, even though catalyt-
  ic activity  was inhibited by prior administration of 2,3,7,8-
  tetrachlorodibenzo-p-dioxin and hexachlorobenzene.

  Hepatoerythropoietic Porphyria
    Hepatoerythropoietic porphyria clinically resembles con-
'  genital erythropoietic  porphyria, presenting in childhood
with severe photo-induced skin damage with inflammation
and scarring associated with increased zinc protoporphyrin
in erythrocytes and acetate-substituted porphyrins in the
plasma, urine, and feces. The principal porphyrin in feces is
isocoproporphyrin (45-47). Erythrocyte uroporphyrinogen
decarboxylase concentrations are less than 10% of normal,
and it is thought that this disease is a homozygous form of
familial PCT. Some authors (21) have reported that immu-
nological measurements of erythrocyte uroporphyrinogen
decarboxyiase are correspondingly decreased, whereas oth-
ers (32) have reported them to be within normal limits. This
could be  due to genetic heterogeneity. The mechanism of
how zinc protoporphyrin accumulates  in erythrocytes is
complex and has been discussed by Elder et al. (45) and Lim
•and Poh-Fitzpatrick (47).

   Few cases of this interesting disease have been reported.
It presents with jaundice and hemolytic anemia at birth (48,
49),  followed in  one case by  the development of mild
photosensitivity at age 11 years. Large amounts of copropor-
phyrin are excreted in the urine and feces, but the pattern of
fecal porphyrin is atypical, the major fraction being hardero-
porphyrin (>60%; normally  <20%). Coprop_orphvrinogen
oxidase activity in the lymphocytes of the patients was 10%
of control values, suggesting  a homozygous  state. The en-
zyme exhibited about 50% of normal activity in both par-
ents. In harderpporphyrin a vinyl group replaces the propic-
 nate group in the A pyrrole ring only. In the normal subject,   •
 the propionate groups in the A and B pyrroles probably are
 converted to vinyl groups in a stepwise manner, oxidation of
 the  group on the A  ring  preceding that on the B ring;
 apparently this process is incomplete in harderoporphyria.

   This disease usually presents in childhood with light-
 induced erythema or urticaria, which is often mild. These
 patients have an increased prevalence of gallstones. Recent-
 ly it has been shown that, hi long-standing cases, there are
 abundant porphyrin deposits in the liver, and liver damage
 and failure may occur (50). The excess erythrocyte protopor-
 phyrin found in this disease is a free unchelated form rather
 than zinc protoporphyrin. The enzymatic defect  appears to
 be a deficiency of ferrochelatase (51). Diagnosis rests on the
 combination of a typical clinical picture and the demonstra-
 tion of increased erythrocyte protoporphyrin concentrations.
 Urinary porphyrin excretion is within normal limits unless
 hepatic cirrhosis impedes biliary porphyrin excretion suffi-
 ciently to increase urinary porphyrin. Two cases of a variant
  of protoporphyria described by Heilmeyer (52) were clinical-
  ly  similar to protoporphyria except that th? erythrocyte
  porphyrin was mostly  coproporphyrin. No fjrther cases
  have been reported.            '
    Erythrocyte zinc protoporphyrin is increased in lead poi-
  soning  and some anemias, including those due  to  iron
  deficiency, excessive blood loss, and chronic disease. When
  insufficient iron is available, or it cannot be readily chelated
  to protoporphyrin, small amounts of zinc protoporphyrin are
  formed and circulate as nonfunctioning hemoglobin (53).

  Acute  Intermittent  Porphyria
    Acute attacks of abdominal pain and neuropsychiatric
   manifestations are the main clinical features of acute inter-
   mittent porphyria;  dermatological lesions do not occur.

               CLINICAL CHEMISTRY, Vol. 32,  No. 7, 1986  1259

 Common features of the acute attacks are: acute abdominal
 pain (in 95%); vomiting (60%); constipation (60%); neuropa-
 thy (60%); tachycardia (60%); hypertension (40%); and men-
 tal changes (50%) (54). About 5% of attacks that are severe
 enough to require hospitalization end fatally, and repeated
 attacks in the same patient are common, so the mortality
 rate is around 15 to 20% (14). Residual paralysis, hyperten-
 sion, and renal failure are long-term sequelae. Acute at-
 tacks are more common in females, more frequently during
 the second and third decades and  are rare  before puberty.
 Attacks are commonly precipitated by drugs, particularly
 barbiturates and sulfonamides, but many other drugs are
 hazardous (11,55). Because of the wide variety of drugs that
 are potentially hazardous  to these patients, it  is more
 advisable to provide the patient with a list  of drugs known
 to be safe rather than a list of the hazardous compounds.
 Endocrine  factors influence the course of the disease, and
 premenstrual attacks are common. Alcohol, infection, and
 fasting are other important precipitants. In  an acute attack
 the diagnosis is made by the association of a typical clinical
 picture together with increased PEG and ALA in the urine.
 Diagnosis between attacks is more  difficult because occa-
 sionally the amount of PEG in the urine is normal (56). If
 this is so and the clinical story  is  convincing, I  usually
 proceed to measure erythrocyte'PBG deaminase.
   The disease is associated with a partial deficiency of the
 enzyme PEG deaminase in several tissues. The  enzyme
 deficiency is inherited as an autosomal dominant trait, but
 only about 10% of the patients who inherit  the  enzyme
 deficiency suffer attacks of the disease (54), so other etiologi-
 cal factors are involved. It is important to identify potential
 or latent cases in relatives of patients with the disease, by
 measuring PEG deaminase in erythrocytes. The amount of
 enzyme  present  in  circulating erythrocytes  is probably
 related to the age and  differentiation  of the cells, and
 therefore measurements are unreliable in  patients with a
 high reticulocyte, count for any reason and in newborns (57).
 There is overlap between the normal and  abnormal refer-
 ence intervals, which limits the usefulness of the test (58).
   Anderson et al. (59) have proposed that clinical expression
 of the disease is  related  to impairment of Sa-reduction of
 steroid hormones by the liver. Glucose (60) and hematin (61)
 are partially effective in treating the acute disease, probably
 by repression of ALA synthetase.

 ALA Dehydratase Deficiency
   This can occur as a primary inherited disorder in which
 the enzyme concentration in erythrocytes is less than 3% of
 normal; it presents as an acute porphyria  (62). Bird et  al.
 (63) have described a family with 22% to 41% of the normal
 concentrations of this enzyme in their erythrocytes;  they
 were asymptomatic. Inheritance  of this latter defect was
 autosomal dominant and may represent the heterozygous
 form of the disease described  by Doss et al. (62). ALA
 dehydratase (PEG synthase) deficiency may also be a conse-
1 quence of lead poisoning. This deficiency may also be seen in
 tyrosinosis, where the abnormal  metabolite succinyl ace-
 tone, present in  excess, competes with ALA (a structural
 analog) for attachment to the active site of the enzyme, and
 patients suffer from acute porphyric symptoms (64).

 Porphyria Variegata
    The clinical features of porphyria variegata are  variable
 and include  acute  attacks and  light-induced cutaneous
lesions. The disease does not usually present before puberty.
The site of the enzyme defect has been argued, but Brenner
and Bloomer (65) have demonstrated that protoporphyrino-'
gen oxidase activity in fibroblasts is 50% of normal in
patients with the disease. Inheritance of the enzyme defect
does not necessarily mean that the patient will develop the
disease; steroid 5a-reduction is commonly impaired in those
in whom the disease has appeared (54). Urinary excretion of
porphyrin precursors and porphyrins commonly increases
during an  acute attack but is usually normal between
attacks. Fecal protoporphyrin and, to a lesser extent, copro-
porphyrin are invariably increased in patients who have
developed the disease, even asymptomatic ones. A homozy-
gous variety of protoporphyrin oxidase deficiency may exist
  McColl et al. (67) have described a family of acute •
porphyries in which some family members have porphyrin
excretion patterns typical of acute intermittent porphyria,
some had patterns typical of porphyria variegata, and 'Still
others had  an intermediate pattern. Only abdominal and
neurological features were present in affected patients; none
experienced photo-induced skin lesions. All had subnormal
activities of PEG deaminase in erythrocytes arid of protopor-
phyrinogen oxidase in leukocytes.

Hereditary Coproporphyria   -
  Clinical features of this disease include acute neurovis-
ceral attacks and photo-induced cutaneous lesions. Sympto-
matic disease does not usually appear before puberty and is
commonly  the  consequence  of drug therapy.  Porphyrin
precursors are commonly increased in the urine during an
acute neurovisceral attack, as is fecal coproporphyrin. Con-
centrations of all can be normal between attapks (68). The-
biochemical defect appears to 'be a partial deficiency of
coproporphyrinogen oxidase activity (50% of normal) in the
tissues (69). Homozygous coproporphyria has been reported

The Effect of  Lead on Heme Synthesis
  Although there has been much argument about the effects
of low concentrations of lead on mental development, overt
symptoms of lead poisoning do not usually appear until the
concentration of lead in the blood reaches 600-800  fig/L
(2.90-3.85 ^tmol/L). Lead appears to inhibit several steps in
the heme synthetic pathway and ALA, coproporphyrin, and
zinc protoporphyrin are produced in excess in the frankly
lead-poisoned patient. Inhibition of ALA dehydratase activi-
ty can be demonstrated at blood lead concentrations as low
as 100-150 /ig/L (0.50-0.70 yomol/L) (71). However, urinary
ALA and coproporphyrin excretion do not increase until the
lead concentration in blood reaches 400 /xg/L (1.90 ^mol/L).
Piomelli et al. (72) have recently reported that erythrocyte
zinc protoporphyrin increases when the blood lead concen-
tration reaches 150-180 /j.gfL (0.70-0.85 jumol/L). The labo-
ratory evaluation of lead toxicity has recently been reviewed
by Fell (73).

Procedure in Investigation of a Suspected Case
of Porphyria
   This has recently been reviewed by With (74),. Moore (75),
and Hindmarsh (76). My approach is outlined in Table 2.
The investigator should determine if the  patient has had
any symptoms of acute porphyria, a history of skin lesions,
and if there is a family history of the disease. If the patient is
 1260  CLINICAL CHEMISTRY, Vol. 32, No. 7,1986

Abdominal pain
(with or without
skin lesions)
Screen for
urinary PEG
Table 2. Investigation of Suspected Porphyria
Result Further action
Negative Excludes acute porphyria as cause of abdominal pain. However, if
you suspect patient is between attacks, quantify urinary PEG and
ALA. If increased, proceed as for positive screen; if normal, mea-
sure fecal porphyrins to exclude VP and HC. If you still suspect
AIP, measure erythrocyte PEG deamihase.
                                            Positive          Confirm by urine PEG and ALA quantification. If clinical picture
                                                            clearly that of AIP, stop here. If not, measure erythrocyte PEG de-
                                                            aminase and fecal copro- and protoporphyrin to exclude AIP and
                                                            differentiate VP, HC.

                                            Normal          Excludes protoporphyria: if strong clinical suspicion, exclude other
                                                            cutaneous porpnyrias by testing urine and feces.

                                            Increased        Protoporphyria confirmed by excluding zinc protoporphyrin.

                                            Negative         Examine feces and erythrocytes for excess porphyrin. If both nor-
                                                            mal, porphyria unlikely.

                                            Positive          Determine porphyrin profile of urine and feces to differentiate PCT,
                                                            VP, ana riC. Measure erythrocyte porphyrin if CEP suspected.
  AIP. acute intermittent porphyria; VP, porphyria variegate; HC, hereditary coproporphyria; CEP, congenital erytnroooietic porphyria; PCT, porphyria cutanea
  Reproduced by kind permission of Clinical Biochemistry.
urticaria or
Skin lesions:
erosions ± bullae,

 suspected of currently having an acute attack of porphyria
 (severe acute abdominal pain, or neuropsychiatric manifes-
 tations, or both), the investigation is easy, because results of
 the Watson-Schwartz urine test will be positive for PEG.
 Many drugs, including chlorpromazine, produce false-posi-
 tive reactions to this test, however, and I usually proceed
 directly to  quantification of PEG and ALA in urine  by
 column chroinatography. If values for these are normal, the
 patient cannot be having an acute attack of porphyria.
   If the patient is between acute attacks, however, it may
 not be possible to establish a diagnosis of "acute porphyria
 between attacks." Values for PEG and ALA in urine are
 commonly normal between attacks in porphyria variegata
 and hereditary coproporphyria and occasionally are normal
 in acute intermittent porphyria. In porphyria variegata,
 fecal protoporphyrin excretion, will be above normal,, even
 between attacks.  Hereditary coproporphyria is very  rare;
 values  for fecal coproporphyrin are usually increased be-
 tween attacks, but may be normal. In acute intermittent
 porphyria, values for erythrocyte PEG deaminase will be
 decreased.  .,
   If the patient has a history of skin lesions, the investigator
 should determine whether these were the urticarial and (or)
 erythematous lesions of protoporphyria or the bullae and
 scars of congenital erythropoietic porphyria, porphyria cu-
 tanea tarda, porphyria variegata,  or hereditary copropor-
. phyria.  A diagnosis of protoporphyria is made from the
 association of a typical clinical picture-with increased proto-
 porphyrin concentrations in the  erythrocytes. PCT can
 usually be 'diagnosed  on the basis of a clinical history of
 alcohol overindulgence, use of the contraceptive pill, and
 increased urinary porphyrins, with uroporphyrin exceeding
 coproporphyrin. If there is no history of precipitating factors,
 it may be necessary to identify excessive isocoproporphyrin
 excretion in feces by thin-layer chromatography. Porphyria
 variegata and hereditary coproporphyria are differentiated
 by demonstrating increased concentrations of protoporphy-
 rin or  coproporphyrin. (respectively) in feces.  Congenital
 erythropoietic porphyria is very rare and is diagnosed by its
 typical clinical presentation and biochemical features.
                                                           Analytical Methods for Porphyrins and Porphyrin

                                                             Methods of analysis for porphyrins and porphyrin precur-
                                                           sors have recently been reviewed by Moore (75) and Bissel
                                                           (77). Normal ranges are method-dependent. Although sol-
                                                           vent-extraction methods can be used for the investigation of
                                                           most cases of porphyria, they lack specificity, and "high-
                                                           pressure" liquid-partition chromatography of porphyrins is
                                                           preferable for those laboratories with this facility, ion-
                                                           exchange chromatography remains the method of choice for
                                                           PEG and ALA.
                                                             "High-pressure" h'quid-chromatographic methods for por-
                                                           phyrin analysis have evolved in several steps. Early work-
                                                           ers used ultraviolet-visible or fluorometric detectors  with
                                                           isocratic or gradient elution of methyl esters on  a normal-
                                                           phase silica column (78). This gives acceptable results, but
                                                           esterification may be incomplete and possibly may alter the
                                                           porphyrin pattern (79). The use of a reversed-phase column
                                                           obviates the need to form esters, and gradient elution gives
                                                           good and fast separations (80). Eiboflavin interference is a
                                                           problem if an ultraviolet-visible detector is used; however, a
                                                           fluorescence detector gives more accurate separations, with
                                                           good sensitivity.
                                                             Porphyrin metabolism  has fascinated chemists since the
                                                           turn of the century and new porphyries  continue  to be
                                                           described. An interesting new development is the use of
                                                           porphyrins for the localization and photodestruction of tu-
                                                           mors (81).

                                                             I am grateful to Dr. S. L. Perkins, Dr. A. Sorisky, Dr.  T. G. Jones,
                                                           Professor S. French, and Mrs. N. Meagher for advice and help
                                                           during the preparation of this review, and to Miss M. Linton for
                                                           typing the manuscript.

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 33. Kushner JP, Edwards CQ, Dadone MM, Skolnick MH. Hetero-
 zygosity for HLA-linked hemochromatosis as a likely cause of tbe
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 34  Mukeiji SK,  Pimstone  NR, Burns  M. Dual mechanism of
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  43  Goldsman CI, Taylor JS. Porphyria cutanea tarda and bullous
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   44 Elder GH, Sheppard DM. Immunoreactive uroporphyrinogen
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                                                                             CLINICAL CHEMISTRY, Vol. 32, No. 7, 1986  1263

                              ENVIRONMENTAL RESEARCH 41, 23-28 (1986)
      ** ,?**,'
       Histopathoiogical Changes Induced in Rat Tissues
                    by Oral Intake of Lead Acetate

               N. KARMAKAR,' R. SAXENA,* ANDS. ANAND!
 Centre for Atmospheric and Fluid's Sciences. Indian Institute of Technology, Delhi Hauz Khas,
    New Delhi 110016; 'University College of Medical Sciences, Ring Road. New Dell,,: and
     tCentrefor Biomedical Engineering. Indian Institute of Technology. Delhi Ham Khax,
                             New Delhi 110016. India
                              Received July 23, 1984

     In the  present study an attempt has been made to observe the pathological alterations
    brought about in the intestine, liver, and kidney of lead-intoxicated rats. A short-term expo-
    sure to a  sublethal dose of lead (44 mg/kg body wt/day) is, seen to cause conspicuous degen-
    erative changes in the three tissues. The intestinal mucosal epithelium is  affected wh.ch
    leads to malabsorption. while in the kidney proximal tubular cells degenerate causing secre-
    tion of essential materials such as glucose, amino acids, etc.. in the urine.  © ivtb Acadcm.c
    Press. Inc.

  Heavy metals in varying concentrations pollute our ecosystem.  Lead is one of
the heavy metals commonly encountered in the environment. Lead accumulates
in the body of man and animals, through water, food, and soil, up to critical toxic
levels It further acts as a  cumulative poison when taken in  small doses and pro-
duces toxic effects (Harrison et al., 1971). A number of studies have been done
on the effect of lead  intoxication on the  ultrastructure of kidneys (Chang et al.,
1981- Beaver, 1961). However, the gut is the organ through which ingested lead is
absorbed into the body and  about 10% of the ingested lead  is absorbed (Rabmo-
witz et al., 1975). Further it is evident from  studies of Wapnir et al. (1977)  that
lead uptake by the mucosa definitely impairs the uptake of glucose, certain amino
acids, and  other nutrients  through the gastrointestinal mucosa. In our preliminary
•experiments, rats were exposed to 2% lead acetate (Pb ac) in food and'the blood
lead level in these rats was 37jxg/100 ml. This level is supposed  to be within safe
limits, but the villi in  the intestine were flattened. Hence in the present case,
experimental studies with lead-treated animals were conducted in order to deter-
mine the extent of histopathological changes produced in the tissues of the small
intestine, liver, and kidney, due to a  short-term exposure to a sublethal dose of
load. The  rat has been chosen as an animal model for lead intoxication and oral
administration of lead  via gavage has been employed as the method of exposure.
Ligl t microscopy was performed on each organ studied and  scanning.electron
micrographs were studied in the  case of the intestine.

   1 Present address: Department of Aeronautics. Physiological Flow Studies Unit, Imperial College.
 Prince Conson Road, London SW7 2AZ. U.K.

                        MATERIAL AND METHODS
  Male and female rats of the albino Wistar strain weighing 250-300 g were used
for the experiment. They were divided into groups of five. All rats received stan-
dard pelleted laboratory diets and were given ad libitum access to drinking water
Group A served as the control. The other groups B, C, and D were exposed to Pb
acetate for varied  durations of time, viz.,^15, and 30 days, respectively. The
method of exposure used was oral administration of Pb ac dissolved m distilled
water  Each  rat  of the treatment  groups  B, C, and D received, via gavage^l
mg/kg body wt/day of lead  as 0.053 M'lead acetate. The control rats received an
equal volume of saline.                                        .
 -  Tissues (small intestine, liver, and kidney) were fixed for historical examina-
tion by light microscopy in buffered Formalin, dehydrated in graded alcohol
series, cleared in  xylene, an'd embedded in paraffin wax. Six-micron-thick sec-
tions were cut and stained with  hematoxylin and eosm.                  •
   Tissue samples  for scanning electron microscopy were fixed in 4% glutaralde-
hyde in 0.1 M cacodylate buffer for 18 hr at 4°C. The fixative was then washed out
of the tissue by three washes in 0.1 M cacodylate buffer and further dehydrated m
araded series of alcohol. Then they were subject  to critical-point drying and fi-
'naily coated with silver. The scanning was done with Cambridge Sterecscan

   During the first  2 days of lead exposure average  food uptake in the lead-treated
 group was 6 g per animal  while that in the control  group was 10  g per animal.
 However  later on there was  no such difference. The earlier discrepancy  was .
 probably merely a taste problem. Further, no significant difference was observed
 in the -rowth of the experimental rats from that of the control group. The histo-
 pathological changes observed at different exposures are given below.

 (/)  Results of the Histological Study after 9 Days Exposure to Lead Acetate
   (a) Small intestine. In two of five cases, mild blunting of the villi was observed.
 The rest of the cases did not differ from the normal.
   (b) Liver  All cases showed sinusoidal dilatation and congestion and one case
 showed presence of extramedullary erythropoiesis. In one case amsonucleosis
 was also observed.
   - (c) Kidney. All the cases were found to be normal.  -

 (2) Results of the Histological Study after 15 Days Exposure to Lead Acetate
    (a) Small intestine.  All the slides showed broadening of the mucosal  villi. In
 one case only,  mild depletion in the number of mucus-secreting goblet cells was
 seen. The broadening of the villi was confirmed in a scanning electron micrograpu

 (F?b) L/ver In one case the changes were found to be within normal limits. In all
  others, centrizonal dilatation and congestion had occurred and hepatocytes were
  vacuolated. One case showed .ballooning degeneration of the liver.        -
    (c) Kidney. Changes in two cases were found to be  within normal limits. In one


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                                    FIG. 1. Scanning electron micrograph of small intestine from a rat sacrificed on Day 15; arrows
                                  show broadening of the tip of villi (x 2000). •
case, the cortical region showed focal areas of .   _
Bother case there were focal areas of tubular degeneration in the medulla	
sional hyaline casts were seen in the tubules.

(3) Results of the Histological Study after 30. Days Exposure to Lead Acetate
  (a) Small intestine. All cases  showed stunted and broadened mucosal villi. In
one of them the villus/crypt ratio had decreased and the v,H. were at the tips  and
at some pies, degenerated. Yet another case showed decrease ,n the number of

S°(bWZ.S Alfcases showed centrizonal dilatation and congestion. Two of them
showed presence of extramedullary erythropoiesis (Fig. 3) as well. In one of them
focal fatty changes had also occurred in patches
  (c)  Kidney. Two cases depicted changes within the normal limits. Two cases
showed focal areas of extreme proximal tubular degeneration dep.cted  as  vac-
uoles  in the medulla (Fig. 4). The rest of the tubules showed severe vacuo a
degeneration which is an indication of  hypokalemia. Occasions hyaline casts
were observed.
 :j ^^^^•'^'•^V^^A^^.A:                                      DISCUSSION
'* •"':::.>'^.V:K-'i£-i        The sensitivity of the active transport mechanism of the small intestinal  mu-
•-•*' r?-&$f£W£.t#*'$      cosa to lead toxlcity has been reported  (Wapnir et al.. 1979). Glucose mtesUna
  •' *    "••*- *"•'•""•  " ••••'* •-•'•      absorption is  considerably reduced in lead-treated rats. Intestinal transport of

                      KARMAKAR, SAXENA, AND ANAND
 • no. 2.  Photomicrograph of sma. intestine fronva rat sacrificed on Day 30; arrows show broaden-
ing of the lips of villi ( x UK) H and H).

some amino acids such a, lysine. glycine, and phenylalanine are also inhibited.
ThereTalo a drastic reduction of intestinal sodium absorpt.on (Wapn.r « aL,

19The histopathological changes observed in the mucosa of the small intestine «.
the present study suggest  malabsorption. Thus they confirm the above findmgs
   FIG. 3. Photomicrograph of liver from a rat sacrificed on Day 30; arrow shows.focus of erythro-
  poiesis in the liver parenchyma ( x40Q H and E).

                                                   LEAD TOXICITY AND RAT TISSUES

'".•*; V -,' ^-:"'l:' ^'; /;^:.:
 Tro'TT^micrograph of renal medulla from a ra« sacrificed on Day 30: arrows show marked
focal tubular degeneration (x400 H and E).

 hat lead causes impairment of active transport mechanisms by adversely af- ,
fecting the mucosa through which the  ions, glucose, etc., are transported from
the lumen of the intestine to the serosa and blood                    int^Tin,i
  Blair ei al (1979) suggest that the interaction between lead and the intestinal
tissue is an adsorption of lead onto the mucosal surface, probably due to its inter-
action with H2PO; present in the glycocalyx of the mucosal membrane by'enzy-
matic hydrolysis of ATP and the formation of lead phosphate.  Due to the infrac-
tion of the lead ions and the mucosa, pathological changes are.caused in the epi-

''""con^picuous degenerative changes were seen in the liver which probably affect
its enzyme activities. The sinusoidal dilatation and congestion is usually a ter-
minal feature due to anoxia. It may also occur due to passive venous congestion: •
The fatty change in the liver is caused by malnutrition and can be correlated to
malabsorption. It has been studied by other workers that the lead content of the  .
liver remains significantly higher than other organs because it is the  mam detoxi-

!>'Lead'toxicity can induce two types of nephropathy. In the case  of acute poi-
soning, as in the present study, proximal tubular morphologic changes have been
observed which cause tubular malfunctioning. Presence of hyaline casts suggests
that protein is being secreted through  the tubules because  premeability of base-
ment membrane of the glomerulus may have been affected. Hypokalemia leads to
 vacuolar degeneration.

_0                       »V^1X..». V1VJ ViV, -J,1,V1_.1. K, . L. ,,^ ........ ~                             ^

  Thus it is seen that short-term exposure to a high, dose of lead manifests  its    <
effect in the form of structural damage at the cellular level.
  It is also concluded that with oral administration of lead acetate the gastrointes-
tinal tract is the primary target organ and shows  pathological changes much be-
fore any other organ.
  Further experimental and mathematical studies  involving the  toxic effect of
lead on the peristaltic transport of the gut have been undertaken  and the results
have been reported in a separate communication.'The effect of lead on red blood
corpuscles is also being studied.
  N. Karmakar expresses her gratitude to Prof. M. P. Singh  for his constant encouragement
throughout this work. The technical assistance of Mr. N. C. Bhatt is acknowledged. The financial    \
assistance from NCERT is also gratefully acknowledged.                                         ;

Beaver, D. L. (1961). The ultrastructure of the kidney in lead intoxication with particular reference to .
    intranuclear inclusions. Amer. J. Pathol. 39, 195-208.
Blair, I. A., Coleman, I. P. L., and Hilburn, M. E. (1979). The transport of the lead calion across the
    intestinal membrane. J. Physiol. 286, 343-350.
Chang, L. W., Wade, P. R., and Lee.  C. W. (i981). An ultrastructural revaluation of lead  induced
    pathology in the kidney. Environ. Res. 26, 136-151.
Goldstein. J.  I., and Yakowitz, H. (Eds.) (1975). In "Practical Scanning Electron Microscopy: Elec-
    tron and  Ion Microprobe Analysis." Plenum. New York/London.
Harrison, P.  R., Matson. W. R., and  Winchester. J.  W.  (1971). Annas. Environ. 5,  613. Cited in
    Hasan, M. Z., Seth, T. D., and Sharma, R. (1974). Determination of cadmium, cobalt, copper.
    lead,  manganese, nickel, and zinc in animal feed by atomic absorption spectrophotometry. Clic-
    mosphere 5,241-246.
Rabinowitz, M., Wetherill, G., and Kopple, J. (1975).  Absorption, storage, and excretion of lead by
    normal humans. In "Trace Substances in Environmental Health-IX" (D. D. Hemphill. Ed.), pp.
    361-376. Univ. of Missouri Press,  Columbia.
Wapnir, R. A., Exeni, R. A., McVicar, M.. and Lifshitz, F. (1977). Experimental lead  poisoning and
    intestinal transport of glucose, amino acids, and sodium. Pediatr. Res. 11, 153.
Wapnir, R. A., Moak, S. A., Lifshitz, R, and Teicherg, S. (1979). Alterations of intestinal and renal
    functions in rats after intraperitoneal injections of lead acetate. J. Lab. din. Med. 94, 144—151.

                                                                                ( itrnczl and laboratory ubsvrvalions     147
    '/  .««,:t, X, U.T.-:.-..V,A ?. LvrCr *KM. t.Hcvo. uf dt^pjn
       j-»i •.:;•. ^-.-i r-gi4il> A ijuiMiid-
       Si.c jiutf; ;.-.' rs3c-.«-s .>;.ti p!si:.T3 Wttr:»'.»-j'.'fi. Ada Nru.-'ji
       StavJ &I:J7&. JV:0'
   !0,  M-.-rxr::; VJ .  P.-:-,.;;.,  N, Ti/jinvni rj.  [{.cj.'i E. Bchcc>!c G.
      .S'.rj.-ijcn SM. Scrcui f". Dia/.quni elimination in premature
      ;::id  full  tcnn infant:, and children. J  Pcrinat Mcd 1:133
   II. i.jr.j:sicl  A.  Mcbcrs A.-Brcdcscn JJ:, Lundc PKM: Plasma
      ccm-cntraiion.-. of Jidrcpum and N-dcsmcihyldinzcpam  in
      :ic»l>orn infants after imravrnotts, intramuscular, rectal and
      oral -administration.. Ada Paediatr Scand 67:699.  I97H.

           Reversible acute  renal failure  in  lead poisoning

           Abdyl J.  Khan, M.D., UrmiJla Palel, M.D., Mohammed Rafceq, M.D.,
           Alfred Myerson, M.D.,  Kasum Kumar, M.D., and Hugh E. Evans, M.D.
           Brooklyn. A'. Y.
  Ai.TMOt:fiii Tm; an i:cis or LEAU on [ho central  ner-
  vous and hcmalopoictic  systems  dominate  the clinical
  picture of lend poisoning, rc-.al manifestations may occa-
  sionally be present.1-3 Kidney impairment also may be a
  side effect of the dictating agents used  in the therapy.3'5
  Renal failure, however, is rare. We describe a patient with
  severe acute renal failure associated with either chelation
  therapy  or preexisting  lead  ncphropathy, or both,  and
  further complicated  by glucose-6-phosphatc dehydrogc-
  nase (G-6-PD)  deficiency.
   A 26-momh-old black girl was admitted lo Jewish Hospital and
 Medical Center of Brooklyn  with  anorexia,  vomiting. lethargy,
 and pallor of one week's deration. Her birth  history and growth
 and development were normal. On rouline screening three weeks
 earlier at another hospital, elevated blood lead level (97 jtg/dl
 whole blood) and free crythrocyte protoporphyrin concentrations
 (1.026 pg/dl crythrocytcs) were found. She was hospitalized there
 and given calcium disodium cdetatc (EDTA)  150 mg and dimcr-
 cnprol (BAL) 48 mg, each intramuscularly every four hours for
 seven days. Urinalyscs  revealed protcinuria before, during, and
 after therapy. Blood urea nitrogen level was 22 mg/dl before and
 after therapy (Table). Scrum crcatininc concentration v.-a;  uot
 measured. At a follow-up visit at that  hospital two weeks later,
 blood lead concentration was 32 ^g/dl whole  blood. The parents
 had noted anorexia and intermittent vomiting since discharge, and
 progressive pallor and lethargy.
   Physical  examination  on admission at Jewish Hospit-1  and
 Medical Center revealed a pale, mildly dehydrated child with

 Fnmi Iht Dtpariment of Pediatrics. Jewish Hospital and Medical
 (.'filter. Sliilf University of New York.  Downstate Medical Cen-
Reprint address: Abdul J. Khan. ,U.O.. Department of Pediatrics.
SSS Prospect PL Brooklyn. i\T 112J8.
  normal pulse, respiration,  and temperature. Blood pressure was
  92/64 mm 1 Ig. Her wi;;»ht  was 24.5 kg, and height 90 cm (surface
  area 0.8 m!). Physical examination was normal. On admission the
  complete  blood count revealed hcmatocrit  22.1%, reticulocytes
  0.3%, platelets 340,000/mm3, and leukocytes 20,400/mm!, with a
  normal differential count. The red blood cell indices indices (mean
  corpuscular volume 66 ^ and mean corpuscular hemoglobin 21
  Wig) and scrum Terrain concentration 8 Ag/dl (normal 15 to 300)
  indicated  iron-deficiency anemia.  Basophilic  stippling was not
  seen and  a  sickle cell  preparation  was negative.  Urinalysis
  revealed proteinuria (3+)  and 2  to 3 red blood cells per high-
  power field (Table). BUN  was 113 mg/dl, and scrum crcatininc
  concentration 13.4 mg/dl.  In view of the severe azotcmia (renal
  failure), further  management included complete  restriction of
  protein  and appropriate  intravenous fluid administration. Urine
 output was adequate during the firsl 24 hours (2 ml/kg/hr), and
 oliguria  did not  occur  subsequently. Renal  function  studies,
 including creatininc clearance and  fractional excretion of sodium,
 were monitored serially and showed gradual improvement (Ta-
 ble). Because the  hematocrit level declined to 17% on day  5, a
 packed  red blood  cell transftision  was given, raising il to 30%.
 Bleeding and coagulation  profiles were normal except for G-6-PD
 deficiency.  Hemoglobin   clcclrophorcsis revealed AA  pattern.
 Results of  scrum  electrolyte (Na. K.  Ca,  phosphorus), comple-
 ment, total protein, and  albumin  studies were normal, i'rotcin
 clcctrophorcsis and immunoglobulins (Ig'G, IgA. IgM), intrave-
 nous pyciogram. renal scan, and  sonogram  also were normal.
 Serum haptoglobin concentration was  227'mg/dl (normal 25 to
 ISO mg/dl). and C-rcactive  protein was -f I. The initial crythro-
 cyte sedimentation  rate was  elevated (90 mm/hr). but gradually
 returned  ro normal. Blood lead  level on admission was 21  Mg/dl
 whole blood, and free  crylhrocytc protoporphyrin concentration
 was 570 Aig/dl crythrocytcs.  The scrum cholesterol concentration
 was elevated (290 mg/dl) on admission, but declined to 225 mg/dl
after .1  week. Blood pressure  fluctuated  between  92/62 and
 110/70 mm Hg. The patient was discharged after 17 days. Two
weeks later,  results of urinalysis were normal. BUN was 19 mc/dl.
«XI2:-.«76/S3/OIOI47+OJSOO.JO/0© 198.1 The C. V. Mosbv Co.

                                                                                               The Jv.trnj! uf Pediatrics

: ilt"**'
' . ri;

/- ^"'-' ."_.'/:;
• /'l/.'/r

; rr.,rf,».

, |
£.'«•*.• i
! 1
' A/?( / i //"O'fc*'*
Jlrt^r i /'///•' i H'tlC/Plll-' tmg/M
' !
. ......
ants Zft'A
'/lir) i>ni;/til)

I'rcri'M: ihc.'.'JpV
Be.'cr^ therapy \*
..'.-/ lrfr,'iip> 35
Alter therapy
»i3 indicates renal injury.
 serum crcatininc 0.7 mg/dl. and hem;itocrit 33%. Twenty months
 after discharge the patient was found to be in excellent health,
 witli normal urinalysis and renal function tests.

   This patient had acute renal failure with reversibility
 over a 12-day interval. Various measurements of renal
 function, -including urine-to-plasma ratios  of creatininc
 and urea and fractional excretion of sodium,  indicated a
 renal  parenchyma! involvement or intrinsic nephropathy
 rather than prerenal azotemia.6-7 Biochemical abnormali-
 ties on admission also included two of three criteria of
 nephrotic syndrome, namely proteinuria >40 mg/m2/hr
 and serum cholesterol >250 mg/dl, but hypoalbuminemia
• was absent. Further evidence of renal parenchymal injury
 included elevated acute-phase reactants (positive C-reative
 protein and elevated) erythrocyte sedimentation rate).
   The cause of renal injury could not be established, but
 chelation therapy must have played a major role. The dose
 of EDTA given to this patient was excessive, and duration
 of therapy was also prolonged (recommended -dose  for
 asymptomatic patients with  elevated blood lead level is
 1,000 mg/M2/day for three to  five days8; this  patient was
 given 1,125  mg/M2/day for'seven days). Maintenance of
 adequate hydration orally or intravenously during therapy
 is helpful in preventing renal injury. Fluids were adminis-
 tered by mouth but not intravenously, perhaps because the
 patient was not toxic, and not  vomiting, during chelation
 therapy.  She did develop evidence of dehydration at one
 point  (urine specific  gravity  1.035) during therapy.
Although EDTA toxicity seemed to have played a major
role, therapy with BAL might also have contributed to the
pathogenesis of renal failure  by  causing  intravascular -
hemolysis in the presence of G-6-PD deficiency.8 The dose
per 24  hours  (360 mg/M2) did not exceed  the recom-
mended  daily dose of 500  mg/M2, but the duration of
therapy (seven days) was longer thari recommended (five
days).8 Although the hematocrit level did fall as a result of
hemolysis, other features of acute hemolytic anemia, such
as high reticulocyte count and  depletion of haptoglobin,
wee absent.
  The possibility of preexisting renal injury caused by lead
itself cannot be excluded. The presence of mildly elevated
BUN (22 mg/d!)  and proteinuria and pyuria  prior to
treatment indicates the  possibility  of lead  nephropathy.
The manifestations of early lead nephropathy are usually
mild and transient and include abnormalities in urinalysis
in 25%  to 30%'-' and amino aciduria in 14% of patients.'
This patient's urine was not examined for amino acids.
Whether  chronic  lead poisoning in  childhood  leads to
chronic renal failure later in life is unknown. In a retro-
spective study in Australia of 401 children admitted during
1915 to 1935 for plumbism, about 25% died of renal failure
and nephritis before reaching 40 years of age.10 In  1962
another retrospective study of 165 children in the United
States did not confirm this high incidence of nephropathy;
only one patient died about 20 years later with "chronic
nephritis"  of  unknown  cause." A kidney  biopsy was
deferred in our patient  in  view of the  rapid  improve-

                                                                             Clinical and lal»)ralory observations     149
                                               C.'-, , t tt —i./
                                               •• *t*r. i ,s~.,Jf


         I ? ^
                  02 D
-) j

    This case emphasizes the need for caution in planning a
 course of therapy for childhood lead poisoning. Initial and
 serial evaluations of renal function (urinalysis, BUN, and
 scrum crcatimnc) are needed. The decision to include BAL
 in the therapy should also be guided  by the  presence  or
 absence of G-6-PD deficiency, as BAL can cause intravas-
 cular hcmolysis in the presence of such deficiency.


    :. PtioclicI SM. Kapilo L. Sciiwachman  H:  Children with an
      incrca.vxl lead burxlcn:  A icrccnin^ and follow-up. JAMA
      Z22:Sb2. 1972.
     . Cli!.x)im JJ  Jr: Aninioaciduria as » manifcsiation of renal
      tubular injury in  lead intoxication and a  comparison with
      pattern of air.niaciduria  seen in other  diseases. J-HeoiATR
      «H17. 1962.  '
     . Chiiolni JJ Jr: Increased lead absorption and lead poisoning
      (p!u:nbi»rn). //, Vaughan VC, McKay  RJ.  Behrman RE.
      editors: Nt-lion text of pediatrics, cd II. Philadelphia. 1979,
      \\ B Saundcrs. p 2025.
     . i-'uivnun H. Pinncgan C. Lushaugh CC: Nepholoxic hazard
      from uncontrolled edathamil calcium-disodium  therapy.
      JAMA  160:1042, 1956.
    . Kcubcr MD, Bradley JE: Acute versenate nephrosisoccurrinj;
      a-, a result of treatment oflcad intoxication. JAMA 174:263,
    . Sclirier  RW: Acute renal failure. Kidney Int 15:205, 1979.
    . Ingullingvr JK, Avner E:  Renal disorders. In Gracf JW, Cone
      Tli, editors: Manual of pcdiatric therapeutics. Boston, 1980,
      Little Brown p 205.
      Chisolm JJ Jr: The use of chclating agents in the treatment of
      acute and chronic lead intoxication in childhood, j PHDIATR
      73:1, 1968.
      Smith HD: Pediatric lead poisoning. Arch Environ Health
     8:68. 1964.
      Henderson DA: A follow-up of cases of plumbism in children.
     Aust Ann Med 3:219,  1954.   '
     Tepper LB: Renal function subsequent  to childhood plum-
     bism. Arch Environ Health 7:82, 1962.
          Lithium  therapy in childhood neutropenia

          Pedro A. de Alarcon, M.D., Jack Goldberg, M.D., Douglas  A. Nelson, M.D., and
          James A. Stockman III, M.D. Syracuse, N.Y.
 NEUTROPHILIA  has been observed  in  most  patients
 treated with lithium carbonate for psychiatric disorders.1
 Because of this  observation, lithium carbonate has  been
 employed in the treatment of neutropenic states, such as
 those associated with rheumatoid arthritis,1 chemothera-
 py,3 and congenital neutropenia.4 The benefits of this form
 of therapy have been variable.
  We report three children  with  chronic neutropenia of
 childhood treated with lithium  carbonate. The neuirophil

 From the Departments of Pediatrics.  Medicine, and Pathology. .
 Upstate Medical Center, Slate University of New York.
 Reprint address: Pedro A. de Atarcon.  M.D.. Department of
 Pediatrics. The Mary Imogenc Basset! Hospital. Coonersiown
 response of these psticnts is evaluated in vivo and in vitro
 using double-layer agar culture techniques.
       CSA       Colony:stimulating activity
       C FU: GM   Colony-forming aniss'--granulceytcs
                  and macrophages
  Three patients (two boys, one girl) with chronic neutro-
pcuia, were evaluated. They were 18 months, 24 months,
and 6 years of age, respectively. The diagnosis was based
on persistent neutropenia with absolute neutrophil counts
of <  1,500 cells/^l and  recurrent infections without any.
other discernible cause for the neutropenia.
  Their  clinical course  was  classified according to the
0022-3476/83/OIOI49-f04S00.40/0 © 1983 The C. V. Mosby Co.

                       Toxicology and Industrial Health, Vol. 8, No. 112,1992   89


                   * Veterans Administration Medical  Center
           Sepulveda  and  University  of  California  School of Medicine
                            Los  Angeles,  California

         t Center for Internal, Occupational  and  Toxicological  Medicine
                            Los  Angeles,  California

                  ^Department  of Physiology and  Biophysics
             University of  Southern  California School  of Medicine
                            Los  Angeles,.  California

      Chronic lead exposure may cause hypertension in normotensive rats. This
      hypertensinogenic effect has been attributed to perturbations in the renin-
      angiotensin axis, the contractile response  of the vascular smooth muscle,
      or the intracellular Ca2+homeostasis as a consequence of the inhibition of
      Na+-K+-ATPase activity. In this study we examined the short-term effect
      of lead exposure on blood pressure, plasma renin activity, vascular con-
      tractility, and renal Na+-K+-ATPase activity and abundance in the sponta-
      neousfy hypertensive rat. Our data indicate that modest lead exposure
  •    caused blood pressure elevation within two weeks in this rat strain that is
      genetically susceptible to the development of hypertension.  This rapid
      blood pressure-elevating effect did not appear to depend on the mecha-
      nisms described in hypertension associated with more chronic lead expo-
      sure listed above. This acute model provides an additional approach to the
      study of lead-induced hypertension.


Epidemiological evidence in humans and experimental studies in animals suggest that
lead exposure may cause an elevation in  blood pressure (BP)  (Sharp et al., 1987).

    1. Address all correspondence to: Nachman Brautbar, M.D., Center for Internal, Occupational
and  Toxicological Medicine, 2222 Ocean View Avenue, Suite #100, Los Angeles, CA 90057.
    2. Key Words: lead, hypertension, spontaneously  hypertensive  rat.
    3. Abbreviations: BP,  blood pressure; Pb-SHR, lead-treated spontaneously hypertensive rat;
PRA, plasma renin activity; SHR, spontaneously  hypertensive rat; TNS, transmural  nerve stimula-
                  Toxicology and Industrial Health, 8:1/2, pp. 89-102
                  Copyright 1992 Princeton Scientific Publishing Co., Inc,
                  ISSN:  0748-2337

90  Nakhoul et al.
Three potential pathogenetic mechanisms for this lead-induced hypertension have been
studied mainly in the rat. These include the effect of lead exposure on the activity of
S renm-angiotensin axis (Fleisher et al, 1980; Victery et al, 1982a)  on vascular ten-
sion and contractility (Webb et al,  1981), and on intracellular Ca2+ homeostasis
(Blaustein 1977- Pounds, 1984; Rosen and Pound, 1989). Special interest has been
directed toward characterizing the inhibitory effect of lead on Na+-K+-ATPase actmty
?Fox et al  1991; Kramer et al, 1986; Moreau et al, 1988; Weiler et al, 1988) which
in turn, would reduce Ca2+ efflux via Ca2+-Na+ exchanger and hence cause increases
in cystolic Ca activity (Pounds,  1984).

Most of these studies measured the effect of chronic lead exposure (>3-7 months) on
the postulated pathogenic mechanisms. It is not clear whether hypertension also devel-
ops following short-term exposure, and if so, whether alterations demonstrated in
 chronic lead exposure are also operative in early phases of lead-induced hypertension.
 In addition virtually all studies have been conducted in rats with no genetic predisposi-
 tion to hypertension. It is possible that the BP-elevating effect of lead is exacerbated or
 potentiated in hosts .that are genetically susceptible to the development of hypertensioa
 In the current study we examined the effect of short-term (3 and 8 weeks), modest lead
 exposure (100 ppm in drinking water) on BP, plasma  renin activity, vascular contrac-
 tility, and renal Na+-K+-ATPase activity and abundance in the spontaneously hyper-
 tensive rat (SHR).

 Animal and Treatment Schedule
 Male 6- to 7-week-old SHR weighing 100-120g (Harlen-Sprague-Dawley Indiana)
 were first acclimatized in hanging  cages, and fed Purina rat chow ad lib (Ralston
 Purina, St. Louis, MO) and distilled water for 3 days. An average weight of' 150g[was
 attdned at the beginning of the study (see weight at  day "0", Figures 2 and 4). They
 were then transferred into individual metabolic cages. Eight ^^/f^Tnm
 the same feeding and drinking  schedule while 8 experimental rats had lead acetate (100
 ppm) added to their drinking water for three weeks (3-wk study). In addition an eight-
  week study was conducted using 7 control and 8 experimental rats (8-wk study).

  Body Weight and Blood Pressure Measurements
  Bodv weight was recorded twice a  week, prior to the measurement of tail artery sys-
'  tolicBP using a tail sphygmomanometer (Friedman and Freed, 1949). Rats were pre-
  wamied for 10-15 min on a heating pad with a surface temperature of 40°C and  were
  then transferred to a temperature-controlled restraining cage where BP was measured
  without anaesthesia. Each measurement represented the  average of 5 consecutive
   readings, approximately 30 seconds apart. The sensitivity of any given reading was 4

                       Toxicology and Industrial Health, Vol. 8, No. 112,1992  91

 Blood, Urine, and Tissue Collection
 Blood was collected at sacrifice at the end of 3-wk in the first study and 8-wk in the
 second study. Trunk blood was collected after decapitation between 7:30 and 10:00
 A.M. For plasma renin activity (PRA), blood was collected into ethylenediaminete-
 traacetic acid and plasma was separated and stored at -10°C until assayed. In the 3-wk
 study, blood for PRA was also collected at the end of the first week by tail-tip amputa-
 tion. Serum creatinine, sodium, potassium, and lead were measured at the end of the 8-
 wk study. A 24-hr urine collection was completed just prior to sacrifice in the 8-wk
 study  for analysis of protein, lead, calcium, and magnesium.  Kidneys were  also
 rapidly excised and stored at -70°C pending assay for Na+-K+-ATPase activity and

 Femoral Artery Vascular Reactivity
 At the end of the 8-wk study femoral arteries were rapidly isolated and cleaned of loose
 fat and connective tissue. Contractile responses of 4mm arterial ring segments were
 measured in vitro using a special assembly (Golub et al., 1989) modified after that of
 Bevan and Osher (1972). The contractile response to transmural nerve stimulation
 (TNS) was obtained following 2 hours of equilibration at 2 gm passive tension. Each
 preparation was stimulated for 30-second intervals with increasing frequencies (1,2,
 4> 8, 16, 24, and 40 Hz) of constant voltage pulses (17 volts, 0.5 msecs duration).
 These  stimulation parameters produced contractions which were totally blocked by
 tetrodotoxin (10-6M, Sigma, St. .Louis, MO) confirming that smooth muscle was not
 directly stimulated. A cumulative dose-response curve to  exogenous norepinephrine
 (1 x lO-8 to 6.5 x 10-5M) was also obtained. All vascular segments were subsequently
 incubated in low potassium (0.6 mM) Krebs-bicarbonate buffer for 15 min, then
 precontracted with norepinephrine (5 x 10-7M). After these contractions reached a
 plateau (5 min), cumulative relaxation responses were obtained by increasing the bath
 KC1 concentration in 0.2-0.4 mM increments, until no further tension changes were
 obtained. At the conclusion of each experiment the arterial segment was again stabilized
 in Krebs-bicarbonate buffer with normal potassium (4.7 mM), and the maximal
 contraction in 100 mM KC1 was measured. Vascular segments were dried overnight at
 100°C and weighed on an electronic microbalance (Cahn Model 26, Torrance, CA).
 Contractile responses to TNS and norepinephrine were expressed as a percent of the
 agonist's maximal response. Cumulative relaxation responses to the readdition of KC1
 were expressed as  a percent of the norepinephrine response in 0.6 mM KC1. Dose-
 response EDso (agonist concentration which produces a 50% of maximal contraction
 or relaxation) was calculated from a linear regression of 15-85% response range and
 compared utilizing Student's t test.

Assay Methods
 Serum creatinine, sodium, potassium,  urine protein, calcium, and magnesium were
 measured using standard laboratory procedures. Serum and urine lead were.measured
 using atomic absorption spectrophotometry (SmithKline Bio-Science Laboratories,

92  Nakhoul et al.
Van Nuys, CA). PRA was assayed as described in prior publications (Eggena et al.,
1991). Renal cortical and medullary Na+ -K> -ATPase activity and abundance were
assessed in crude microsomes prepared as described by Schmitt and McDonough
(1986).  Activity was measured as ouabain-sensitive inorganic phosphate liberation
from ATP and expressed as umol phosphate per mg protein per hour (Lo et al., 1976).
Protein  concentration was measured by the method of Lowry et al. (1951), using
bovine serum albumin as the standard Immunoblot analysis of Na+ -K+ -ATPase
subunit  abundance was carried out using a modification of the method reported by
Renart et al. (1979). Briefly, 25 ug homogenate protein was resolved by SDS-PAGE
and electrophoretically blotted onto diazophenylthioether (DPT) paper. The DPT paper
was incubated with a combination of rat anti-a monoclonal antibody (McKl diluted
1:200, from K. Sweadner, Harvard Medical School) and anti-Bi antiserum (diluted
1:100, from R. Leverson, Yale Medical School).

Statistical Treatment of Data
All data were presented as mean±SEM. Student's t-test was used for determination of
significant difference between variables, using a standard statistics package (Winer,
1962).           '  .

Blood Pressure and Body Weight
In the 3-wk study, both the control and the experimental SHR demonstrated steady BP
for the first 8  days (Figure 1). On days 12 and 14, systolic BP rose in the lead-treated
SHR, but not in the control SHR. Thereafter, the BP in both groups exhibited incre-
mental rises, but with the lead-treated rats displaying significantly higher systolic BP
than the control rats for all measurements. Figure 2 depicts the progressive weight gain
in the two groups of rats. No difference was observed between the control and the
lead-treated rats.
                  O - O =Control Group
                  • - • =Lead Group
                                8       12   14
FIGURE 1. Systolic blood pressure in control and lead-treated rats in the 3-wk study.

                                       Toxicology and Industrial Health, Vol. 8, No. 112,1992  93




150 -
O 	 O =Control Group ' I
• 	 • =Lead Group •
/ O
T //\
-x*^<»-"*" 	 II
L^^>T J 1
. •*
                                    04        8       12  14    17     20
                          FIGURE 2. Body weight of control and lead-treated rats in the 3-wk study.







O — O =Control Group
• — • =Lead Group T
*=p<0.001 i^^ *
* * t \/ ^•'^
* * I-'*"
* r T^^

/ 0 0 Q
T/ /i^?"9 9 ?
•^* X"
T T /o — O— o
i JL^— — ^o*^"'i i -^


— a

                                 03    8111417   22263033384145   51    58
                      FIGURE 3. Systolic blood pressure in control and lead-treated rats in the 8-wk study.

                Figure 3 summarizes the BP changes in the second study, confirming the observation
                of significantly higher BP in the lead-treated SHR versus control SHR by the end of
                the second week. BP in the control SHR also rose during the third week, however, the
                lead-treated SHR had higher BP throughout the remainder of the  8-wk (58 days)
                study. In this study, the lead-treated group demonstrated  significantly higher body
                weight from day 30 through the end of the study (Figure 4).

94   Nakhouletal.

Blood and Urine Chemistry
The blood level in 7 rats after 8-wk lead treatment was 5.3±3.0 (range 4-8) mcg/dl, or

2.5 (range 1.9 to 3.9) x 10-7M. The blood level in all 7 control rats was less than 2

mcg/dl, the lower detection limit. Urine lead concentration and 24-hr urine volume and

lead excretion on the last day of the 8-wk study are depicted in Figure 5. Compared to

control rats, all lead-treated'rats had higher urine lead concentration, urine volume, and

daily lead excretion. Serum concentrations of creatinine, sodium, potassium, and 24-hr

calcium and magnesium  did not differ between the control and the lead-exposed rats

(Table 1>. Neither treatment group had measurable protein in the urine.

       'S  280-


       ^  260-1

        £2  240-
                  O	O=Control Group
                  •	• =Lead Group

^ 220-
0 200-


m^~ /
t Jw r\

J$ •!•
< 7
n J , — , 	 1 — <—

                  03    8 111417  22  263033 3841 45   51    58

                                         DAYS •

           FIGURE 4. Body weight of control and lead-treated rats in the 8-wk study.
150i '


                       Toxicology and Industrial Health, Vol. 8, No. 1/2,1992  95
 Plasma Renin Activities (PRA)
 Figure 6 summarizes the PRA in the control and experiment rats in both the 3-wk and
 the 8-wk studies. The 1 week measurements (Figure 6A) were based on blood col-
 lected by tail-tip amputation in the 3-wk study, and the 3 week (Figure 6B) and the 8
 week (Figure 6C) measurements were based on trunk blood collected at the end of each
 of the two studies, respectively. PRA was not different between the control and the
 lead-treated rats at all three time-points.

 TABLE  1.    Serum and Urine Chemistry Parameters in  the 8-Wk Study

Mean= 0.41
SE= O.O6

Na* mmol/l
Cont Lead
15O 154
148 142
1 53 1 53'
156 149
163 148
1 49 1 46

1 53 1 48
2 2
Cont Lead
6.3 6.4
8.O 8.1
8.3 8.8
8.6 7.7
'8.7 7.8"
7.3 7.9

7.8 7.7
O.4 O.3
Cont Lead
O.14 O.12
O.O8 O.1O
0.08 O.13
0.10 O.O8
O.O6 O.1O
O.14 O.16
O.OO 'O.I 5
. 0.13 O.O8
0.09 O.1 1
O.O1 O.01
TABLE 2.    Effect of  Lead-Exposure on Vascular  Contractility and
               Relaxation In Vitro

               Procedure (N)

                                 Maximal Response, gms
KCI, (lOOmM)
Control (6)
Lead (8)

Control (7)
Lead (8)

Control (5)
Lead (6)
 6.2 ± 1.1 (Hz)
 7.7 ± 1.1 (Hz)

3.4 ± 0.1 (10-7M)
3.8 ±0.1 (10-7M)
1.4± 0.4
1.7 ± 0.3

3.3 ± 0.5
3.9 ± 0.5

1:8 ± 0.3
2.1 ± 0.3

               Procedure           EPfp. mM K+	Maximal Response, gms
Control (6)
Lead (8)
0.5 ± 0.1
0.5 ± 0.1
Femoral Artery Contractility
The effect of lead treatment for 8 weeks on various indices of femoral artery vascular
responsiveness are summarized in Table 2. The EDso to both TNS and norepinephrine

96   Nakhoul et al.
was not different between the lead-treated and the control rats, suggesting that lead
treatment did not alter vascular sensitivity to norepinephrine, either from TNS-induced
endogenous release or from an exogenous source. The lead-treated SHR did exhibit an
apparent lower response to TNS, but the difference was significant only at 8 Hz (lead,
n=8 45±4% versus control, n=6, 55±3%, p<0.05). Maximal contractile response to
TNS, norepinephrine, or 100 MM potassium also did not differ between the lead-
treated and control vascular preparations (Table 2 A). In addition, the sensitivity (EDso)
and the maximal relaxation response to potassium-induced vascular relaxation were
also similar in the two treatment groups (Table 2B). The maximal relaxation of both
lead-treated and control groups to the readdition of potassium, expressed as a percent
of the norepinephrine response in low potassium, was similar (lead 106±3 versus con-
trol 110±4%)' Finally, there was no significant difference in the dry vessel weights
between both groups (lead-treated 0.210±0.005 versus control 0.210±0.010 mg. per 4
mm segment).                       30]iwEEK      T    ^^
                                       3 WEEKS
 FIGURE  6. Plasma renin activity
 (PRA) in control and lead-treated rats.
 I WEEK. Measurements on tail blood
 at the end of one week in the 3-wk
 study. 3 WEEKS. Measurements on
 trunk blood at the end of 3-wk study.
 8  WEEKS. Measurements on trunk
 blood at the end of the 8-wk study.
                                       8 WEEKS
  Renal Na+ -K+ -ATPase Abundance and Activity
  Na+ -K> -ATPase a and B subunit abundance measured in crude membrane from renal
  cortex and medulla'of the 3-wk study was not different between control and lead-

                       Toxicology and Industrial Health, Vol. 8, No. 112,1992  97

 treated rats (Figure 7). Renal cortical and medullary Na+ -K+ -ATPase activities, mea-
 sured at 3- and 8-wk were also not different between control and lead-treated rats.


 With the exception of Aviv et ail (1980), who observed BP increases after 6 weeks of
 lead treatment, all other studies were conducted following 3-7 months lead exposure
 (lannacone et al., 1981; Victery, 1988). These studies used normotensive rats not pre-
 disposed to the development of hypertension. Our study suggests that in SHR, lead
 exposure causes accelerated BP elevation within two weeks. Thus, genetic predisposi-
 tion to hypertension may constitute one factor for the proposed heterogeneity in host-
 susceptibility to lead toxicity (Raghvan, et al., 1980). We have no explanation for the
 higher body weight in SHR exposed to lead for more than 30 days (Figure 4). This
 increase in body weight did not appear to be important for the observed increase in BP
 which'manifested itself between days 12 and 17 in both the 3-wk and the 8-wk study.
 The increase in urine volume in the absence of weight loss suggests the possibility of
 increased water consumption in the lead-treated rats.
                                      C  C  Pb Pb
                    C  C  Pb Pb
FIGURE 7. Immunoblot analysis of
Na+-K+-ATPase  cc and 6 subunit
abundance in renal cortex and medulla
from control (C) and lead-treated (Pb)
rats.  Analysis carried out using 25ug
homogenate protein per lane, probed
with  I125-labeled anti-a and anti-Bj
Two other studies on the effect of lead exposure in SHR have been reported. Evis et al.
(1987) studied the-effect of chronic lead exposure (3 months) on experimentally
induced cardiac arrhythmias in post-weaning (4-week-old) SHR. They noted that at 5
weeks, SHR which drank water containing 250 or 1,000 ppm lead had marked and
significantly higher BP than SHR which drank lead-free water. No mention was made
concerning whether  BP was measured prior to  5 weeks. In contrast, Wiecek et al.
(1986), studying 12-week-old (100-150g) male SHR treated for 10 weeks with 0, 0.5,
0.1, and 1.0% lead acetate in drinking water (0, 250, 2,500, and. 5,000 ppm, respec-

98  Nakhoul et al.

lively) observed a BP-lowering effect in a dose-dependent manner. Parenthetically, the
body  weight of the 12-week-old male SHR used was unusually low because the
expected weight would range between 240 and 260g.

The unanticipated observation of Wiecek et al. (1986) may be explained, in part, by the
study of Victery et al. (1982a). These latter authors noted that while 100 ppm of lead in
the drinking water led to the development of hypertension in the male rat, (Sprague-
Dawley, confirmed through personal communication), exposure to higher doses
negated the hypertensinogenic effect (Victery et al., 1982a). Although Victery et al.
(1982a) did not use SHR, our study would confirm that 100 ppm drinking water is
also "hypertensinogenic" in SHR.  This observation, in combination with those of
Wiecek et al. (1986) (whose SHR received 250 ppm or higher doses of lead) would
suggest a biphasic effect of lead on BP in SHR, that is, a BP-elevating effect at 100
ppm and a BP-lowering effect at higher doses. Such a hypothesis, while dovetailing
with the observation of Victery et al. (1982a), is at variance with the observation of
Evis et al. (1987), who documented a clear BP-elevating effect in SHR given 250 and
1,000 ppm lead in the drinking water.

It may be relevant to point out that Wiecek et al. (1986) noted that "despite the absence
of elevated basal BP in surviving Pb-SHR, a high proportion of Pb-exposed SHR died
from cerebral hemorrhage." It was  not clear whether the reported BP was only taken
from the surviving Pb-SHR. It is interesting to note that Evis et al. (1987) also ob-
served that at 8 weeks, 7 of the 8 high-lead-dose  rats died upon warming in preparation
for BP measurements. The reason for this fatal sensitivity to heat and the direct cause
of death were not investigated.

The rapid manifestation of the hypertensinogenic effect of lead in our animals allowed
us  to examine the question of whether the pathogenetic mechanisms believed to be im-
portant in sustaining the hypertension associated with chronic lead exposure are also
 involved in the early phases of lead-induced hypertension. As mentioned earlier, previ-
 ous studies have focused on three major pathogenic mechanisms, i.e., the effect of lead
 exposure on the activity of the renin-angiotensin axis (Fleisher et al., 1980; Keiser et
 al., 1983; Victery et al., 1982a), on vascular tension and contractility (Webb et al.,
 1981), and on Na+ -K+ -ATPase activity and the consequent effect on intracellular
 Ca2+ homeostasis (Blaustein, 1977; Kramer et al., 1986;Weiler et al., 1988).

 Lead exposure has been reported to cause either an increase, no change, or a decrease
 in PRA in the rat, and appears to be influenced by factors such as the dose and duration
 of lead exposure and the age of the animal when exposure first began (Vander, 1988).
 In one-month-old rats whose exposure to lead began in utero, PRA was increased
 compared to age-matched controls (Victery et al., 1983). However, in rats in which
 lead exposure was prolonged 5 to 6 months, either no change or a reduction in PRA

                                      Toxicology and Industrial Health, Vol. 8, No. 1/2,1992  99

                was observed (Victery et al., 1982a). In the chronic exposure studies it was observed
|||             that lead-induced hypertension was associated with low PRA (Victery et al., 1982a;
W             1982b). However, it was postulated that the hypertension may have first gone through
                a high-renin phase during the earlier stages of lead exposure (Victery et al., 1982a;
                1982b). In our study, measurement  of PRA at 1  week and 3 weeks, a  time period
 ;-              bracketing the acceleration of hypertension in the lead-exposed rats,  failed to demon-
                strate such an early, high-renin phase (Figure 6, A and B), suggesting that the rapid
 j:              initiation of lead-induced BP elevation can occur independent of changes in PRA. PRA
:||            " measured at the end of the 8-wk study (Figure 6C) also failed to demonstrate a differ-
 ^              ence between the control and the lead-exposed rats.

jj              Webb and associates (1981) studied the effect of lead exposure on the vascular reactiv-
                iity of the tail artery in the rat. They observed an increase in BP in adult male Wistar rats
                given drinking water containing 100 ppm lead, as lead acetate, for 7 months (147±4
 :              versus 133±4 mmHg in age-matched controls,  p<0.05). In a 1981 study, Webb et al.
                obtained findings that were similar to ours; they noted no difference between the treated
                and control rats in contractile response to high concentrations of extracellular KC1 or to
                electrical-field stimulation of the adrenergic nerve endings. They did demonstrate that at
||,             a given concentration of either methoxamine or norepinephrine the contractile force was
If             greater in arteries form lead-treated rats. The authors suggested that  the lead-induced
£              hypertension was associated with an increased vascular responsiveness to a-adrenergic
J|              agonists. However, neither their study nor the present data demonstrated a difference
if              in sensitivity to adrenergic stimuli (as reflected by ED$Q values). In addition to differ-
j|              ence in rat strain and blood vessel preparation, Webb's group extended lead treatment
'"*              to 7 months, which increased the likelihood of vascular structural adaptations to hyper-
                tension. Our study thus suggests that lead-induced hypertension can occur prior to the
                development of adrenergic hyperresponsiveness.

                Blaustein (1977) and Haddy and Overbeck (1976) have reviewed the importance of the
                Na electrochemical gradient across the sarcolemma in the regulation of vascular smooth
                muscle tension and contractility. For example, an inhibition of Na+ -K+ -ATPase activ-
                ity by a humoral agent such as the natriuretic hormone can lead  to a reduction in the
                Na-gradient directed into the cell, the driving force for extruding intracellular Ca
                through the Na-Ca exchanger. The consequence is an increase in intracellular Ca activ-
                ity and thereby an augmentation in vascular smooth muscle tension  and contractility.
                Kramer et al. (1986) reported a 50% inhibition (150) of Na+ K+ -ATPase activity in rat
                renal cortical homogenate incubated in 7 x 10-5M lead chloride. A subsequent study
                from the same laboratory using purified Na+ -K+  -ATPase from hog cerebral cortex
                also demonstrated an ISQ of 8 x 10-5M for lead (Weiler et al., 1988). In our in vivo
                study the blood lead level attained ranged between 2 to 4 x 10-7M (4 to 8 ug/1), a con-
                centration two orders of magnitude lower than those used in in vitro studies. This may
                explain our failure to demonstrate a change in Na+ -K+ -ATPase activity .and a and 6

100  Nakhouletal.

subunit abundance in either the cortical or the medullary homogenates of kidneys from
lead-treated rats. Fox et al. (1991) also noted that renal Na+ -K+ -ATPase activity in
adult (90 days) rats with blood lead levels of 5-7 ug/dl was not reduced compared to
that found in control rats not previously exposed to lead. Our finding that potassium-
dependent" vascular relaxation was not different between the control and the lead-
exposed rats was also consistent with a lack of an effect of lead treatment on the Na+
K+ -ATPase system in the vascular smooth muscle. Previous vascular contractility
studies have shown that  potassium-induced relaxation of precontracted vascular
smooth muscle  is  ouabain-sensitive and correlates with Na+ -K> -ATPase activity
(Webb and Bohr, 1978). The data taken together would suggest that the effect of lead
on blood pressure is not a direct consequence of changes in the Na-Pump activity or its
pool size.

In conclusion, we found that modest lead exposure used to induce hypertension in
chronic animal studies can  cause rapid BP elevation in rats genetically susceptible to the
development of hypertension. In such early-phase lead exposure the initiation of the
BP elevation effect appears to be independent  of the conventional mechanisms
discussed. This acute lead-induced  hypertension model provides an additional
approach to the study of this important disorder.


This study was  supported in part by funds provided by the Department of Veterans
Affairs and the  Center for Internal, Occupational and Toxicological Medicine, Los
Angeles, California. Drs. Farid and Kayne were supported by fellowship awards from
the American Heart Association,  Greater Los  Angeles Affiliate,  and Dr. Hu was
supported by a research fellowship from the National Kidney Foundation of Southern

AVIV, A., JOHN, E., BERNSTEIN, J., GOLDSMITH, I., and SPITZER, A. (1980). "Lead intoxication
    during development: Its late effect on kidney function and blood  pressure." Kidney Int. 17:
BEVAN, J.A. and OSHER, J.V. (1972). "A direct method for recording tension changes in the wall
    of small blood vessels in vitro." Agent actions 2: 247-260.
BLAUSTEIN, M.P. (1977). "Sodium ions, calcium ions, blood pressure regulation, and hyperten-
    sion: A reassessment and a hypothesis." Am J. Physiology 232: C165-C173.
    CLEGG, K., NAKHOUL, R, and LEE, D.B.N. (1991). "The influence of recombinant human ery-
    thropoietin  on blood pressure, and tissue renin angiotensin system of the rat." Am. J. Physi-
    ology 261 (5) Part 1, pp. E642-646.
EVIS, MJ., DHALIWAL, K., KANE, K.A., MOORE, M.R., and PARRATT, J.R. (1987). "The effects
    of chronic lead treatment and hypertension on the severity of cardiac arrhythmias induced by

                         Toxicology and Industrial Health, Vol. 8, No. 1/2,1992   101
    coronary artery occlusion or by noradrenaline in anaesthetized rats." Arch. Toxicol. 59: 336-
FLEISHER, N., MOUN, D.R., and VANDER, AJ. (1980). "Chronic effects of lead on renin and renal
    sodium excretion." J. Lab. Clin. Med. 95: 759.
FOX, D.A., RUBINSTEIN, S.D., and HSU,  P. (1991). "Developmental lead exposure inhibits adult
    rat retinal, but not kidney  Na,K-ATPase." Toxicology and  Applied Pharmacology 109: 482-
FRIEDMAN, M. and FREED, S.C.  (1949).  "Microphonic manometer for indirect determination of
    systolic blood pressure in the rat." Proc. Soc. Exp. Biol. Med.  70: 670-672.
GOLUB, M.S., LUSTIG, S., BERGER, M., and LEE, D.B.N. (1989). "Altered vascular responses in
    cyclosporin-treated rats." Transplantation 48: 116-118.
HADDY, F.J.  and OVERBECK, H.W. (1976). "The role of humoral agents in volume expanded
    hypertension." Life Science 19: 935-948.
IANNACONE, A., CARMIGANI, M., and BOSCOLO, P. (1981). "Neurogenic and humoral mecha-
    nisms in arterial  hypertension of chronically lead-exposed rats." Med. Lav. 72: 13-21.
KEISER, J.A., VANDER, A.J., and GERMAIN, C.L. (1983). "Clearance of renin in unanaesthetized
    rats: Effects  of chronic lead exposure."  Toxicology and Applied Pharmacology 69: 127-137.
KRAMER, H., GONICK, H., and LU, E. (1986). "In vitro inhibition of Na,K-ATPase by trace
    metals: Relation  to renal and cardiovascular damage." Nephron 44: 329-336.
LO, C.S., AUGUST,  T.R., LIEBERMAN, U.A., and DELMAN,  I.S. (1976). "Dependence of renal
    Na,K-adenosine  triphosphate activity on thyroid status." J. Biol. Chem. 251: 7826-7833.
LOWRY, O.H., ROSEBROUGH, N.J., FAW, A.L., and RANDALL, RJ. (1951). "Protein measure-
    ment with the folin phenol reagent." J.  Biol.  Chem.  193: 256-275.
    B., FESTY,  B., and LEIIOUCH, J. (1988). "Influence of membrane  sodium transport upon the
    relation between blood lead and blood  pressure in a general male population." Environmental
    •Health Perspective 78: 47-51.
POUNDS, J.G. (1984). "Effect of lead intoxication on calcium homeostasis and calcium-mediated
    cell function. A review." Neurotoxicology 5:  295-332.
RAGHVAN, S.R.V., CULVER,  B.D., and GONICK, H.C. (1980). "Erythrocyte lead-binding protein
    after occupational exposure. 1. Relationship to lead toxicity." Environmental  Research  22:
RENART, J., REISER, J., and STARK, G.R. (1979). "Transfer of proteins from  gels to diazobenzy-
    loxymethyl-paper and detection with antisera: A method for studying antibody specificity  and
    antigen structure." Natl. Acad. Sci. 76:  3116-3120.
ROSEN, J.F. and POUNDS, J.G. (1989). "Quantitative interaction  between Pb2+ and Ca2+ home-
    ostasis in  cultured osteoclastic bone-cells." Toxicology and Applied Pharmacology 98: 530-
   . 543.
SCHMITT, C.A.  and  MCDONOUGH, A.A. (1986). "Developmental and thyroid hormone regulation
    of two molecular forms of Na.K-ATPase in brain." J. Biol. Chem. 261:  10439-10444.
SHARP, D.S., BECKER, C.E., and SMITH,  A.H. (1987). "Chronic  low-level lead exposure. Its  role
    in the pathogenesis of hypertension." Medical Toxicology 2: 210-232.
SHIONO,  K.  and SOKABE,  H.  (1976). "Renin-angiotensin system in spontaneously hypertensive
    rats." Am. J. Physiol. 231:  1295-1299.
VANDER, A.J. (1988). "Chronic effects  of lead on the renin-angiotensin system." En •ironmental
    Health Perspective 78: 77-83.
VICTERY, W. (1988). "Evidence for effects of chronic lead exposure on blood pressure in experi-
    mental animals:  An overview. Environmental Health Perspective 78: 71-76.
VICTERY, W., VANDER, A.J., SCHOEPS, P., and GERMAIN, C. (1983). "Plasma renin is increased
    in young rats exposed to  lead in utero and during nursing." Proc. Soc. Biol.  Med.  172: 1-7.
VICTERY, W., VANDER, A.J., SHULAK, J.M., SCHOEPS,  P., and JULIUS, P. (1982a). "Lead,
    hypertension, and the renin-angiotensin system  in rats." J. Lab. Clin. Med. 99: 354.
    (1982b). "Lead exposure begun in utero decreases renin and angiotensin II  in adult rats." Proc.
    Soc. Exp. Biol. Med. 170:  63-67.

102  Nakhouletal.
    effects of lead on vascular reactivity in rats." Am. J. PhysioL 241. H21 1-H216.
WEBB, R.C. and  BOHR, D.F.  (1978). "Potassium-induced relaxation as an indicator of Na -K
    ATPase activity in vascular smooth muscle." Blood vessels 15: 1V5-2U/.
WffiCEK  A  MANN J.F E., NOWACK, R., and EBERHARD, R. (1986). "Effects of lead mtoxica-
    Sn 'on blood ^cnuM and vascular reactivity in spontaneously hypertensive rats." Journal of
                                   -^ GONICK, H. (1988), "Effects of lead and na^uretic
     horrnone on kinetics of sodium-potassium-activated, adenosme tnphosphates: Possible rele-
     vance to hypertension." Environmental Health Perspective 78:^ 113-115.
WINER, B.J. (1962). Statistical Principles in Experimental Design. McGraw-Hill, New York, NY.

          'Richard P. Maas
          Steven C.'Patch
          Diane M. Morgan
          Geoffrey M. Brown

   US EPA Project Manager: Jeff Cohen
        Technical Report #5-019
          DECEMBER 1995



                The National Primary Drinking Water Regulation for Lead ("The Rule") requires many
     -   public water systems to monitor for lead in tap water, and to take various steps (including corrosion
         control, public education, and lead service live line replacement) to reduce lead exposure for their
         customers if the levels are found to be elevated. The Rule applies to "community" water svstems
        '(those which serve at least 25 people year round), and to  "non-transient, non-corrmiunity"' water
       '  systems (serving at least 25 of the same people for at least six months of the year). The Rule
         excludes systems referred to as "transient non-community" water systems. These systems cater
         to transitory customers in non-residential areas such as campgrounds, motels, and gas stations. The
         US EPA excluded transient systems because  adverse health effects' caused by lead hi drinking
        water would generally be associated with long-term cumulative exposures rather than the shon-
       •term exposures predicted from these types of systems.  The purpose of this study was to collect
       • actual data on lead in drinking water from transient systems-in order to better characterize potential
        exposure'risks.        '

              The names and addresses of 8000 transient water systems were supplied to the EQI by the
        US EPA, Office of Groundwater and Drinking Water.  A letter was sent to all 8000 systems
      •  (approximately 800 in each of the ten EPA regions) asking for'their participation in the study in
        return for the EQI  laboratory providing free lead testing of their drinking water.  The letter
        emphasized that no action, regulatory or otherwise, would be-taken contingent on sample results
        and that all individual results would be kept confidential. Two-hundred-eleven request forms were
        returned from the pool of systems. A test kit with laboratory cleaned bottles, sampling instructions
        (Attachment 21), and a research questionnaire (Attachment £2) was sent to'each of these 211
        respondents. Over a three month period, which included two sets of follow-up phone calls, a total
        of 115 systems returned their samples, with these participants then forming the database for the
       survey analysis described herein.

              As noted above, 8000 geographically distributed transient water systems were invited to
       participate in the survey study and thereby receive free confidential lead-testing. The actual survey
       population is a self-selected group based on their commitment to follow through with testing and
       returning samples'and, thus,'may be a biased sample in one respect or another. Table  1 below
    •   shows the number of sites separated by system classification and by their first draw lead levels.
       Although no rigorous statistical analysis of the original potential study population of 8000 sites was
       conducted, the actual 115 respondents appear to represent a wide range of the types of systems
       envisioned for the study. There may be a slight over-representation of governmental buildings (15)
       and park visitor centers (19),.which is probably to be expected because these personnel might be
    •   more apt to participate in a government-sponsored survey study.

                         Lead Levels lnd
           Type of System

    Gas Station
    •'   '     i.-

    Campground Building
    	——	.—,	

    Campground Hookup
   Highway Rest Area
   Ski Resort

   Mobile Home

   Visitor Center
   •••'"      -••  ._

  Government Building

A Mantel.Haenszel Chi-Square test for differences between system types revealed no statistically

Significant differences in the risk of first draw lead contamination as a function of system type (p

       Table 2 summarizes 'first draw lead concentration ranges as a function of the type of-water

dispensing device.  Again, no statistically significant differences were observed between the
various types (p = 0.67).

       Some general descriptive statistics derived from the
Table 3.
                                                    survey data are presented below
TABLE 3. General Descriptive Statistics of Transient System Lead


                                   Quantile Concentrations
            One-Minute Purged Line Lead Quantile Concentrations
	 ; 	 • 	 •
!0'0%(Max> -
50% (Median)
Arithmetic Mean
Percent > 15 '

"' l
                                                 90th P€rcentile ^ draw concentration is ?0 3
                                                  l5u8fL ^— rage fot draw concenidon
   nenn    "                                       *« Upper fa°md of *e ^lean exposure of a
   person  dnnkmg  water from these, transient systems.  The average one-minute  pur^d-L
   concentrate was 2.3, which probably represents a good estimate of the lower boL       "
   visitor exposure at facilities served by transient water systems.  Also, it can be seen
   minute purge reduces the mean lead exposure by approximately 75%.

         It should be noted that -the statistics presented up to this point include plumbing systems
  of all ages rather than just the 1983-I988.age range for Tier 1 residences under the Rule Table 4
  shows the breakdown of first draw lead results by age-of-piumbing systems.
Age Category (Yrs.)
0 --4.9
5.0 - 9.9
• 10-19.9
20+ •
Number of
• Lead Concentration Range (ug/1)
68.8 ...
• 67.6
5-15 '
• 33.3
• 16.7
As can be seen from Table 4, very few facilities had new plumbing systems, and the distribution
of elevated lead levels shows very little dependence on plumbing system age (p = .66). Previous
EQI research using much larger residential .databases have observed this same trend.

       Table 5 shows first draw lead levels as a function of the age of the water dispensing device.
As can be seen from the data the percentage of samples having lead levels above 15 ug/1 was
virtually identical for all three age groups, and no statistically significant association between
dispenser age and.lead concentrations was observed (p = 0.83).

                       Lead Levels by Reported
                                         Lead Concentration Range (ug/1)
TTie statistical association between water standing time in the plumbic

°nm      *™
                                                                       system and
the extent to which visitors to these transient systems might actually be exposed to water
with excessive standing times was beyond the scope of this study. In most cases it seems
likely that only small percentages of visitors would encounter such circumstances
TABLE 6. First Draw Lead Levels by Reported Water sendin
Standing Time
Category (Hrs)
6 -.10
10 - 14
Number of
Concentration Range (ug/1)
-T*> 1
• 73.9
10.0 _ '
17.4 '

 sates M «£ onfstS? * "*? °f "« P'°^' Compassed 35 sta.es. Nine
 Virginia f6), New Yo^fj T in?^ ""l^^^ -P-entation were U^h (U),
 Nebraska (4).                  (  '' M'SSOUn (6)' North Carolina («>• Montana (5), aid
 across tteUs    ,te/QI "^ "*** °V" i0'°°° residences ^ «"™ercial
 ^sient water systems included in this study were almost ail suppiied by welistd^.s

residences as a whole, especially those served by.private weils and springs


 1. Attachments
    . Lead test instructions
2. Table A
     Information on Transients Sites

Attaint             .
                                        Noa-Community Water Systems
   I. Name of system or business

  2. Address
  	.	Phone_
         a. System  number (as shown on address label on mailer)	

  4.  Water source:	well,   supplied by a public water utility	
        	other (please specify)	
  5. ,Do you treat the water in-any way besides chlorination?	.      	

  6. Water system type:    '                         '            •
        	Gas Station          '	Motel/hotel/lodge      	Ski resort
        	Campground building          ' -   Highway rest area	Store
        	Campground hookup        	"Restaurant            	Mobile home park
        	Other (please specify)__	•	_^

 7. Type of water service line from water source to building: 	copper,   	lead    	plastic,
        	galvanized steel,   	combination,  	don't know, • other	
        a. Age of this  water line in years	

 S.  Type of indoor plumbing within building to water outlet:	copper,   	lead,.  	plastic,
              	galvanized steel.  	combination,- 	don't know,  other	
       -a. Age of this water line in years	
        b. Age of building	

 9 . Water sample source: • -        '                                        .
      '	self cpntained water cooler,	water-fountain,  	kitchen faucet, 	bathroom faucet,
       	hookup,  	outdoor spigot
       	other (please describe)^	•	'
       a. Manufacturer of source noted in question 9
       b. Age of source noted in question 9	•
       c. Used primarily by:  	employees,  	transients (customers)

10. Do you have a water filter attached to the line from which this sample was taken?'	
       a. If there is a filter, is it a 	sediment filter only,  	carbon filter, 	cation exchange filter,
              	combination to" remove particles, chlorine and metals
       b. Manufacturer	

II. Do you have a water conditioner attached to the line-from which this sample was taken?	
       a. Manufacturer	__-	

12. Approximate standing time of water before first draw sample was taken (in hours)    •	

13.' Dateofsample__.	

   Attachment #
            .     The Environmental  Quality Institute
                        UNCA "One University Heights "Asheville NC  28804

                Lead Test instructions for Non-Community Water Systems

  Enclosed are two sample bottles for collecting your tap water and a questionnaire with labels.

  First, you must decide which of your taps you want tested. Second, fill in your name and address on the
  labds proved. These labels will identify your samples and will be used as address labels for
                                                                           bdow for

  Procedure 1:  First Draw Sample  use the one liter container              •'             •
  This sample should be taken from the cold water tap sometime when the water has been standing in the
  plumbing lines for at east six hours and before the toilet is flushed or water is run in the building  Fully
  open the enclosed co apsible one-liter plastic container. Place it under the faucet and turn on the cold water
  to a moderate flow Mien it is full, replace the cap.  Dry the outside of the bottle. Immediately attach the
  . t irst Draw  label to the sample bottle.  (Make sure the bottle is dry or the label won't stick well.)

  Procedure 2:  Purged Line  use the small 30 ml bottle but you will need to supply avne quart pitcher
 or container for measuring.                                                rr j      ^    y
 This sample should be taken after water has' been purged through the plumbing system'. Run the cold water
 tap at a high rate for exactly 60 seconds, then reduce the flow to a small trickle.  Place a one-quart or one'- '
 liter contamer (preferably a plastic one, but glass 'is acceptable) under the  faucet  and fill  When the
 container is full, stir the water in it briefly with a plastic (Not Metal) spoon or other stirrer.  Pour water from
 the container into  the small sample bottle.  Dry the outside of bottle.  Immediately attach the "Pur-ed Line"
 label co this bottle.           ,                                                        s

 Be sure to fill out and enclose the questionnaire. Please leave  the label with your address stapled to the
 questionnaire, it will be the mailing label used by the  lab to return your results to you.  This will help ensure
 proper identification of your samples. Place the label saying "Environmental Quality Institute  UNCA,  -
 Asheville. NC 2S804" on the outside of the box and mail to the laboratory within  10 days of'sampling.

 The laboratory results will be sent back to you within two to three weeks of receipt, along with information
 on what the. results mean and whether any further action beyond line purging is recommended.

 If you have any questions about how to take these samples or fill out the forms-please call Diane Morgan at
(704) ZD 1-6800. Your participation in this project will help  determine the  extent of the lead problem at your
facility and in transient, non-community water systems as a whole.

      I	i

March 20, 1996

TO:          Judy Lebowich
             Connie Bosma

FROM:      Jeff Cohen

SUBJECT:   Analysis of UNC-Ashville Survey of Lead at Transient Systems

       This is to summarize results of the UNC-Asheville survey on lead levels in transient
water systems. The survey results are documented in the report, A Survey Study of Lead in
Drinking Water Supplied by Transient Water Systems, Technical Report #95-019, December
1995, prepared by the Environmental Quality Institute of the University of North Carolina at

UNC-Asheville Survey

       Of 8,000 transient systems throughout the country invited to receive free lead testing, 115
participated.  The relatively small number prevents meaningful analysis of the representativeness
of the respondents, although a wide range of system types across the country is included. First
draw-(1-liter) and one-minute purged (30 milliliters) samples were collected at each site.

       While the median and average concentration of the first draw samples were relatively low
(2.3  and 9.2 parts per billion (ppb), respectively), high levels were measured at some of the sites.
Ten percent of the sites had levels in excess of 20.3 ppb, with a maximum first draw sample of
229  ppb.

       The average one-minute purged line sample was 2.3 ppb, with a 90th percentile of 3.4
ppb. The purged samples had much lower concentrations (75% lower on average), and less
variable readings, although two sites had levels above 30 ppb.

       High lead readings were correlated with long standing times and with age of the
plumbing system.  All of the sites with first flush levels above 50 ppb had standing times of six
hours or greater. Some standing times were as high as 96-120 hours, resulting in levels of 34 and
56 ppb, respectively.

       Standing time is not the only explanatory variable for high lead levels, however. The
maximum reading of 228.8 ppb was measured in a mobile home park (self-classified as a
transient system), where the water had been standing for six hours.  The fact that the purged
sample level was only 0.7 ppb raises doubt about the validity of the first flush sample. Assuming
no contamination occurred and it is a legitimate result, the use at the site of a water softener and a
sediment filter (which if not maintained, can build up excessive lead particle concentrations)
could explain the high reading.

       Implications of UNC Study Results

       As is the case with most any survey of lead in drinking water, the study found elevated
lead levels at some locations that appear to be correlated with standing time and plumbing age.
The influence of water corrosivity and other variables was not measured, and the possibility of
sampling errors and contamination cannot be ruled out.

       Given the body's ability to safely "filter" out most lead that is ingested, and the fact that
lead toxicity is typically associated with long-term exposure,1 isolated consumption of water with
even extremely high lead levels (e.g., >100 ppb) would not necessarily constitute a significant

       Assuming the results are accurate and representative of nationwide exposure at transient
systems, the remaining question is whether sensitive groups (women of childbearing age,
pregnant women, and young children and infants) in transient systems may be regularly exposed
to the high lead levels found at,the relatively small percentage of sites in the study. This question
is outside he scope of the UNC-Ashville study and conducting a representative exposure
assessment for sensitive populations in transient systems would be difficult.
       'U.S. Environmental Protection Agency. June 1986. Air Quality Criteria for Lead,
Volume IV. Environmental Criteria and Assessment Office, Research Triangle Park, NC.


                          Health and Ecological Criteria Division
                            Office of Science and Technology
                                    Office of Water


The long-term effects of low level lead exposure are well documented and are the basis of
numerous regulatory programs aimed at reducing lead exposure of all segments of the population
but particularly among children and women of childbearing age. Less is know about the effects
of short-term exposures to low levels of lead, especially among the groups that are most sensitive
to the effects of long-term exposures.

The EPA drinking water lead and copper rule is one of the regulatory programs focused on
reducing environmental lead exposure. This rule was promulgated in 1991 (USEPA, 1991).
When promulgated, transient, noncornmunity water systems1 were excluded from the rule's
requirements based on the fact .that exposures to lead that would occur at such facilities would be
acute rather than chronic.

The decision to exclude the transient noncommunity systems was supported by lexicological
data from studies in adults which identified increased concentrations of erythrocyte
protoporphrine and depressed activity of aminolevulinic acid dehydratase as the critical effects
from short-term lead exposures (Cools et al., 1976; Schlegel and Kufner, 1979; Stuik, 1974 as
cited by Cohen, 1993).  These effects are markers for inhibition of heme synthesis (ATDRS,
1992; Hendmarsh, 1986) but are reversible and do not persist after exposure has ceased.
Aminolevulinic acid dehydratase is the key enzyme regulating the rate of heme synthesis and
erythrocyte protoporphrine is a precursor to heme and, thus, a biomarker for heme production.

Heme is the  oxygen-binding pigment in the red blood cell. Physiological replacement of red
cells is a continuous process. Therefore, the red cells in circulation cover the spectrum of
nascent cells to cells ready to be removed by the spleen for degradation.  Thus, a short term
deficit in heme production is not immediately manifest in a decreased supply of red blood cells.
The gradual  turnover of the red blood cells is one of the factors that protects against acute
hematological effects resulting from a short term lead exposure.  A short term event influencing
heme synthesis will not be have a parallel impact on the number of functional red cells in

A study by Stuik (1974) of the short-term effects of lead on heme synthesis was key in the
       '•Transient noncommunity water systems are public water systems that serve fewer than 25
of the same people at least six months a year.


original EPA decision to exclude transient systems from lead monitoring requirements (Cohen,
1993). As reported hi ATSDR (1992) two groups of 5 women and one group of 5 men were
orally exposed to 0.02 mg/kg/day lead hi the form of lead acetate for 21 days during this study.
This dose is equivalent to 1 mg lead per day for a 50 Kg reference woman and 1.4 mg lead per
day for a 70 kg reference man. Under conditions where water consumption is 2 L/day, this dose
is equivalent to a water concentration of 500 Mg/L for the women and 700 Mg/L for the men.

       0.02 ms/kg/dav x 50 kg = 0.5  mg/L = 500 Mg/L

       0.02 mg/kg/dav x 70 kg = 0.7  mg/L = 700 Mg/L
             2 L/day

In this study, suppression of the activity of erythrocyte aminolevulinic acid dehydratase became
apparent by day 3 of exposure. The degree of suppression increased until  day 14 and then
remained constant for the remainder of the study.  Effects on erythrocyte protoporphrme were
noted hi the women but not the men after 2 weeks of exposure. Blood lead levels had increased
to 40 Mg/dL or higher before effects on erythrocyte protoporphrine were noted.

Unfortunately, no studies were identified that examined the effects of short-term exposure to lead
on heme synthesis in children. Lead absorption in children is greater than that in adults.
Chamberlain et al (1978) as cited in ATSDR (1992) found that children absorbed about 3.3 times
more lead than adults.  Using this relationship one might infer that short- term lead exposures
that were about one third that hi adults (0.006 mg/kg/day) or greater could have an effect on
heme synthesis hi children equivalent to that seen in adults.

       0.02mg/kg/dav = 0.006 mg/kg/day

 For a 10 kg child consuming 1 L of water per day this would be equivalent to a concentration of
60 Mg/L.

       0.006 mg/kg/dav x 10 kg = 0.06 mg/L = 60 Mg/L
             1 L/day


2.1    Human Data

 In order to determine if additional data have become available regarding the effects of short-term
exposure to lead since EPA's original evaluation, EPA conducted a search of the Medline
databases for acute studies published hi the last ten years. Only one article which included
primary data on the effects of short-term exposure to lead in humans was identified.

This article presented a case study of a 26 month old black female who was hospitalized because
of anorexia, vomiting and lethargy (Khan et aL,  1983). The child had been seen three weeks
earlier at another hospital and found to have a blood lead level of 97 ,wg/dL.  At that time she was
treated with calcium disodium ethylene diamine tetraacetic acid (EDTA) and dimercaptol every 4
hours for seven days. She was then discharged to her parents. When she returned to the hospital
2 weeks later her blood lead levels had declined  to 32 /zg/dL.  Three weeks after the original
hospital admission, the child was brought to the  hospital because of the vomiting/anorexia
problem. Blood urea nitrogen (BUN), proteinuria, and serum creatinine values indicated acute
renal failure. The child also was found to suffer from a glucose-6-phosphate dehydrogenase
(G6PD) deficiency.  With treatment the renal problems were resolved and kidney function
normalized allowing the patient to be discharged after 17 days.

The treating physicians attributed the renal problems to the amount of EDTA administered
during chelation therapy (Khan et aL, 1983). They felt that renal effects from the EDTA may
have been complicated by the patient's G6PD deficiency and the dimercaptol therapy. Potential  .
contribution of lead to the renal failure could not be eliminated because of slight proteinuria and
minimal elevation of BUN present at the time of the initial hospital visit. However, the authors
felt that contribution of lead to the renal problems, if any, was minimal.  However the potential
for an adverse effect of lead on renal function should have been considered when initiating the
EDTA treatment.

2.2     Animal Data

Several articles regarding short terms effects of lead on animals were also identified in the
literature search. Studies were classified as acute only if the exposures lasted 14 days or less;
this is the Agency for Toxic Substances and Disease Registry (ATSDR, 1992) classification for
an acute animal study. In evaluating the data from these studies it is important to remember that
they are not directly applicable to humans because of differences in physiology and life-span. A
one week exposure in a rat with a two-year life-span is not equivalent to a one week exposure in
a human with an average 70 year life-span.

Two of the animal studies looked at the effects of lead on renal, function and/ or blood pressure or
related end points. The shortest duration evaluated was 7 days.  Giridhar and Isom (1990)
examined the response of the atrial natriuretic factor (ANF) to intraperitoneal injections of 0.01,
0.1, 0.5 or 1 mg/kg lead acetate solution in groups of 4 Sprague-Dawley rats. ATF is a peptide
hormone that acts as a neuromodulator to control blood pressure and fluid volume. At the end of
7 days, ANF levels decreased in the plasma and hypothalamus.  The response was  roughly
related to dose but the dose-response trends were not consistent across the doses. The ANF
levels in the atria were not dose-related, but were increased as compared to the controls for three
of four doses.

Water ingestion did not change over the period of lead exposure but urine volume decreased in a
dose-related fashion (Giridhar and Isom, 1990).  Increases in body weight suggested that the

decreased urine output was the result of fluid retention and that the fluid retention rather than the
lead was responsible for the lower levels of ANF in plasma.  Since evidence of lead-induced
nephrotoxicity was not evaluated hi this study, it was not possible to conclude whether or not the
effects observed were the impact of the lead on the kidney. The use of intravenously
administered lead also reduces the value of this study for evaluating risk in humans from lead
exposure through drinking water.

In a second study, spontaneously hypertensive Sprague-Dawley rats were given drinking water
containing 100 mg/L (100,000 /ug/L) lead as lead acetate for 3 weeks (Nakhoul et al., 1992).
There was no change in the systolic blood pressure of the animals for the first 8 days of exposure
even in these genetically susceptible rats. However, by day 12 and through day 20, the systolic
pressure of the rats was significantly higher than that for the controls.  These results suggest that
short-term, low-level lead exposures would not have a hypertensive effect in humans even those
with a genetic predisposition to hypertension comparable to that in the rats unless they persisted
for a month or more.

Histological changes induced by lead from lead acetate in intestines, kidney and liver were
evaluated in Sprague Dawley rats by Karmaker et al. (1986).  A dose of 44 mg/kg for durations
of 9, 15 or 30 days was evaluated in groups of 5 rats. This dose is equivalent to 3,080 mg/day in
a 70 kg human or a concentration of 1,540,000 //g/L with ingestion of 2 L of water per day.
After 9 days, mild shortening of the intestinal villi were seen in 2 of 5 rats and histological
changes in the liver were observed in all rats.  No renal abnormalities were observed.  After 15
days, intestinal and liver abnormalities had progressed and affected more animals than at 9 days;
3 of 5 rats showed histological kidney abnormalities. The changes observed were probable given
the magnitude of the dose, even though the exposures were relatively short-term.  Comparable
exposures from drinking water are unlikely even from very corrosive water in systems with lead-
containing components.


An examination of recent data on the effects of short-term lead exposure do not change the
conclusions of the original review by EPA (Cohen, 1993). Effects on heme synthesis  still appear
to be the critical effect from lead exposure.  These effects are first manifest by decreased activity
of aminolevuluiic acid dehydratase within as few as three days and occur at exposures below
those from other lead-induced effects. Concentrations  of between 500 and 700 ^ug/L were
observed to change the enzyme activity in adult humans. Due to differences in lead absorption,
the response in children might occur at an normalized concentration as low as 50//g/L. The
normalized concentration is the average concentration for all water consumed during the day and
is not the same as the "first draw" concentration. Continued ingestion of water with a
normalized concentration of this value for a period beyond several weeks would not be advisable
for children from a health perspective.


ATSDR. Agency for Toxic Substances and Disease Registry. 1992. Toxicological Profile for
Lead. U.S. Department of Health And Human Services. Public Health Service. Agency for Toxic
Substances and Disease Registry. Atlanta, GA. TP-92/12.

Cohen, J. 1993. Memo to Jim Elder of the US Environmental Protection Agency Office of
Ground-water and Drinking Water and an Attachment entitled: Draft Response to NRDC  .
Assertion that Transient Systems be Regulated under Lead NPDWR. July 21.

Giridhar, J. And G.E. Isom. 1990. Interaction of lead acetate with atrial natriuretic factor in rats.
Life Sci. 46(8):569-576.

Hindmarsh. J.T..  1986. The porphyrias: Recent advances. Clinical Chemistry 32 (7): 1255-1263.

Karmakar, N., Saxena, R. and S. Anand.  1986. Histopathological changes induced in rat tissues
by oral intake of lead acetate. Environ. Res. 41(l):23-28.

Khan, A.J. Patel, U., Rafeeq, M, Myerson, A., Kumar, K. and H.E. Evans. 1983. Reversible
acute renal failure in lead poisoning. J. Pediatr. 102 (1):147-149.

Nakhoul, F., Kayne, L.E., Brautbar, N., Hu, M., McDonough, A., Eggena, P., Golub, M.S.,
Berger, M., Chang, C., Jamgotcbian, N., and D.B.N. Lee. 1992. Rapid hypertensinogenic effect
of lead: studies in spontaneously hypertensive rat. Toxicol Ind. Health 8(l-2):89-102.

USEPA. U. S. Environmental Protection Agency. 1991. Maximum contaminant level goals and
national primary drinking water regulations for lead and copper; Final rule. Federal Register.56