United States
                   Environmental Protection
                   Agency
Health Effects
Research Laboratory
Research Triangle Park NC 27711
                   Research and Development
EPA/600/S1-85/009  Aug. 1985
x°/EPA         Project Summary

                   Assessment  of  Cadmium
                   Exposure and Toxicity
                   Risk in  an American
                   Vegetarian Population
                   Rodney E. Willard
                     It has been postulated that non-
                   vegetarians may be exposed to less
                   cadmium than vegetarians because of
                   the cadmium-poor meat in their diet.
                   This study attempts to test this possi-
                   bility by measuring the cadmium expo-
                   sure and accumulation in a population
                   subgroup that includes many vegetari-
                   ans. The conclusions are: 1. no statisti-
                   cal difference in cadmium exposure
                   rate is demonstrated between the non-
                   vegetarians and lacto-ovo-vegetarians
                   of this study; 2. the cadmium exposure
                   rate of the small group of pure vegetar-
                   ians studied is in the direction antici-
                   pated (higher than nonvegetarians) but
                   is not large enough to achieve statisti-
                   cal significance; 3. cadmium accumula-
                   tion in the critical organ (kidney) of sub-
                   jects coming to autopsy is well below
                   the range associated with renal injury;
                   and 4. quantitative  methods for mea-
                   suring total urine protein are not as sat-
                   isfactory for assessing tubular protein-
                   uria as those developed to measure the
                   small proteins that normally appear in
                   the glomerular filtrate.
                     This Project Summary was devel-
                   oped by EPA's Health Effects Research
                   Laboratory, Research Triangle Park, NC,
                   to announce key findings of the re-
                   search project that is fully documented
                   in a separate report of  the same title
                   (see Project Report ordering informa-
                   tion at back).

                   Introduction
                     Only 3% of the cadmium used in the
                   United States is recycled. The remain-
der is dispersed into the biosphere
where it may contaminate our drinking
water  and enter the food chain. Esti-
mates based on autopsy data and envi-
ronmental analysis have led to the fear
that lifetime exposure and accumula-
tion may be nearing a critical toxic level
in the  general population. Schroeder's
animal experiments and several au-
topsy series have suggested a relation
between unrecognized  cadmium accu-
mulation and hypertension. Up to 20%
of the total body burden in  smokers
may be related to  absorption from
tobacco-born cadmium. Analysis of
foods from cadmium-contaminated re-
gions shows variation in cadmium con-
tent. Cereals and other foods of  plant
origin  contain  higher  concentrations
than most foods of animal origin. Re-
gardless of whether cadmium enters
the  body via the gastrointestinal tract
from food  and water or the respiratory
tract via smoke or dust, the physiologic
biochemical pathways result in its even-
tual binding to  metallothionein. This
5000 dalton protein has the greatest
binding affinity for cadmium of any pro-
tein studied thus far and easily carries
the cadmium  with it through the
glomerular basement membrane into
the  renal tubular filtrate. The normal
tubular function  for conservation  of
amino acids and proteins results in the
removal of cadmium-metallothionein
complex from the filtrate into the tubu-
lar mucosa. The cadmium remains se-
questered in the renal cortex for the rest
of the life of the individual. When a suf-

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ficient concentration has accumulated,
renal tubular dysfunction with cad-
miumuria, beta-2-microglobuluria, and
Fanconi syndrome become apparent.
Additional accumulation  leads to de-
struction of the renal cortex. The
scarred contracted end stage kidney is
indistinguishable from that of glomeru-
lonephritis, pylonephritis, or hyperten-
sive  renal disease.
  It  has  been postulated that  non-
vegetarians  may be exposed to  less
cadmium than vegetarians because of
the cadmium-poor meat  in their diet.
The food  animal is exposed to the same
water and plant sources as the human
and  the cadmium accumulates  in his
kidneys by similar physiologic mecha-
nisms as in the human.  As a conse-
quence the skeletal muscle is depleted
of its cadmium. Steak is cadmium-poor;
sweet breads, especially kidneys and
liver, are not. Food derived from skeletal
muscle will dilute the cadmium ob-
tained from the cadmium-accumulating
food plants.
  This study attempts to test this possi-
bility by measuring the cadmium expo-
sure and  accumulation in a population
subgroup that includes many vegetari-
ans. The study group is drawn  from
Southern California Seventh-Day Ad-
ventists (SDA) already enrolled  in the
ongoing Adventist  Health Study.
Seventh-Day Adventists are a small
Protestant denomination with  about
three million  members worldwide. Ap-
proximately  100,000 live  in California.
Church standards require that members
abstain from the use of tobacco and al-
coholic beverages. Previous studies
have shown  that over 98% of church
members conform to  these standards.
The  church also recommends other
practices affecting life  style that are not
used as criteria for membership. A veg-
etarian diet is urged as the ideal. The
use  of unclean  meats as  biblically de-
fined (pork, shellfish, etc.) is proscribed.
Use  of caffeine-contained beverages
(coffee, tea, colas) and  strong condi-
ments (such as black pepper) is discour-
aged. Whole grains, nuts, vegetables
and fruits as major dietary elements are
recommended. A food industry special-
izing in protein products  derived from
plant sources has developed. Although
these dietary  practices have  been
taught for over 100 years, members
vary widely  in actual  practice. A very
few  are  pure vegetarians or vegans
(PV). About half  are lacto-ovo-
vegetarians (LV), who use eggs and
dairy products but abstain from meat,
poultry and  fish. The remaining non-
vegetarians (NV) use clean meats and
fish in their diet. About 17% drink one or
more cups of coffee per day. Epidemio-
logic studies among this group began a
quarter of a century ago in the content
of unhealthful effects  of tobacco usage.
These led to the prospective Adventist
Health Study begun in  the 1970's. Ap-
proximately  half of the California Ad-
ventist population  are  enrolled.  They
completed life style questionnaires and
respond periodically to health status in-
quiries. Hospital  record and death cer-
tificate information supplements health
data obtained directly from the partici-
pants.
  The public concern for identifying and
minimizing environmental health risks
mandates a monitoring  of exposure
rates. Following  index population
groups that  have higher than average
exposure to cadmium may be useful in
providing warning  of general popula-
tion health risk.
Participants

Live Subjects
  From the computerized records of the
Adventist Health Study, 50 pairs con-
sisting of one SDA lacto-ovo-vegetarian
and one SDA non-vegetarian were gen-
erated. The members of each pair were
matched by age, sex, marital status, ed-
ucation,  menopausal status, and occu-
pational  group. Since cadmium accu-
mulates over the life span of the
individual at a biological half life of ap-
proximately  20 years, these subjects
were selected from the 40- to 60-year-
old age group. Another matching  was
attempted to develop a similiar pairing
with 50 pure vegetarians. Because these
vegans are such a small  fraction  of
those enrolled in the Adventist  Health
Study, the age requirements were re-
laxed to  increase the possibility of re-
cruiting a complete group.  These sub-
jects  were identified  only by a
transformed identification number. The
ID  numbers of the  subjects that
matched were passed  back to a com-
puter routine that  mailed letters ex-
plaining the study to subjects. Each sub-
ject was randomly assigned to  one  of
the two  dieticians for  the  rest  of the
study. The dietician was given a name,
address, and phone number. Any fur-
ther information the dietician received
about the subject came directly from the
subject.  If a subject declined to  partici-
pate, none of our staff learned anything
further about the subject, even whether
or not he was part of the Adventist
Health Study.
  Five to seven days after the introduc-
tory letter  was  mailed, the dietician
called the participant. She briefly ex-
plained the study, answered any ques-
tions, and made an appointment for the
first home visit. If the subject agreed to
participate,  a letter confirming the ap-
pointment and an introductory manual
briefly explaining specimen collection
was mailed. At the first interview, a writ-
ten consent was obtained and a detailed
explanation of the collection procedure
made both verbally and in the form of
an extensive instruction manual.
  When a subject declined to partici-
pate, a computer routine was used in an
attempt to replace him. If he was a veg-
etarian subject the computer searched
the files for another match. If this  was
unsuccessful, the dietician assigned to
contact the  matching  subject was in-
structed to  cancel the appointment for
home visit  with  the matching subject,
and expressed our regrets. If another
match was found, and home visits with
the original subject had not been sched-
uled,  they were  postponed until a few
days after the new subject had received
the introductory letter.
  Once the  first home visit had been
completed,  all subjects were carried
through the completion of sample col-
lection, regardless of withdrawals of
matched subjects. The first visit  was
conducted  by a research nutritionist.
Any questions at this point were an-
swered. Written  consent was  obtained
from the study subject. Then the subject
was asked  to  recall  food intake in the
previous 24 hours, using  models  and
following a prod question sheet.  The
purpose of the unannounced recall was
to determine  if  the subjects  changed
their diets while completing the three-
day diary.  Seventh-Day Adventists in
particular may have a number of ideas
about  an appropriate  diet which they
may  not be following exactly.  The
knowledge  that their diet is being ana-
lyzed may cause them to consciously or
unconsciously change their eating.
  A health  history (exclusion  criteria)
questionnaire was administered by the
nutritionist. This questionnaire included
inquiries about a number of characteris-
tics that were the basis for further exclu-
sions. These included osteomas, major
changes in diet in the last two years,
and use of a number of drugs, including
cancer  chemotherapeutic agents and.
lipid lowering  agents. The use of estro-'

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gens was recorded, but not used as
grounds for exclusion.
  The subject was instructed in the col-
lection  of urine and feces. A  2.5-liter
container and  a 1-liter widemouthed
plastic jar in  an airline bag  were pro-
vided for 24-hour urine collected on the
second  day of feces collection. This ex-
cluded the first  voiding of that day and
included the first voiding of the follow-
ing day. The container  provided con-
tained a preservative  (HC1). The fecal
samples included  every stool during
three days. Plastic  bags were provided
to hang in  the  toilet, or a port-a-potty
made available  if so desired. After defe-
cation, the bag was removed, deposited
in another plastic bag and the outer bag
closed,  and placed in a freezer  chest
packed with dry ice. This outer bag was
labeled  with the subject's identification
number, the  date, whether contami-
nated with urine, and a sequential num-
ber to identify  the bowel movement.
Subjects were instructed to urinate be-
fore defecating to minimize urinary con-
tamination of  the fecal sample. The
plastic bag was hung  in the toilet  in
such a way that there was some  space
in front of the bag for males to urinate.
In case of females, a small amount  of
urine might fall  into the bag. There also
was space beind the bag to drop  paper
into the toilet.  A small checklist was
used to identify any stools with urinary
contamination. Employed subjects
were provided with another freezer
chest at their place of employment if
they felt it likely they would defecate at
work. This was  labeled "Biological Ma-
terials for Scientific Research—Do Not
Disturb or Remove." Our staff contacted
the employer to deliver and pick up the
chest.
  All subjects were asked to collect food
samples. All foods and beverages (ex-
cept water) consumed were weighed on
a gram  scale. During the three days of
fecal collection, the subject was asked
to place about 2 ounces of food or bev-
erage other than water, in a small plas-
tic container labeled with the names of
the food. Dressings, sauces, etc., added
at the table were collected separately,
as these may not  be  mixed homoge-
neously on the plate with the foods with
which they are eaten. A number of small
containers, each containing 2 ounces of
a different food, could be  placed in a
larger container  labeled for the data and
the meal. The food samples were refrig-
erated, but not frozen. Vitamin pills and
other dietary supplements were col-
lected in exact duplication of the
 amount used by  the  subject, and  a
 record of the  nutritional  supplement's
 composition obtained by the dietician.
  Each subject was given forms to
 record his complete dietary intake dur-
 ing the three days of fecal collection. He
 was instructed to  record all food and
 beverages consumed,  and to save
 recipes of homemade foods.
  The  subject was given a day within
 the following next week to begin his di-
 etary  record  and sample collection.
 Each collection period included one
 weekend day; that is, it was Thursday,
 Friday, and Saturday, or Sunday, Mon-
 day, and Tuesday.
  The  24-hour recall  was coded by the
 dietician for  the dietary analysis the
 same day if possible.
  On the  second day of  the collection
 period, the same dietician phoned the
 subject to review the procedures and
 answer any questions. This allowed dif-
 ficulties to be handled early. Specific
 questions were asked to ascertain com-
 prehension and compliance.
  On the workday following the sample
 collection (that is, a Monday or  a
 Wednesday), the dietician retrieved the
 freezer chest containing the fecal and
 urine samples, another containing the
 food samples, and when used, a third
 containing samples from the subject's
 place of employment.
  The  dietician reviewed  the three-day
 diary with the subject  and edited the
 diary as necessary.
  The  subject's height, weight, triceps
 skinfold and blood pressure were mea-
 sured.
  The  dietician checked each food cup
 for sample adequacy and completeness
 and reviewed the urine and fecal sam-
 ples collected for completeness.
  The  same day the  dietary diary was
 coded  whenever possible.
  The  dietary interviewer made an ap-
 pointment for  a venipuncturist to draw
 blood, and explain the 12-hourfast. This
 appointment was made as soon as pos-
 sible after the period of food collection,
 within  no more than seven days. It was
 not made during the food collection pe-
 riod, or on the day following, to prevent
 the  fast from  interrupting the subject's
 usual eating habits.
  The  venipuncturist visited the sub-
ject's home, usually early in the morn-
 ing  to  draw the blood samples for the
 biochemical  profiling. Those subjects
 living within easy driving distance from
the  medical center were  scheduled to
 have the blood work drawn in the out-
 patient venipuncture station.
 Autopsy Subjects
   Kidney, liver, pancreas and hair sam-
 ples collected by cooperating patholo-
 gists in Southern California Seventh-
 Day Adventist hospitals were obtained
 from subjects coming  to autopsy. Life
 style and  health data were obtained
 from hospital charts,  next-of-kin, and
 from the Adventist Health Study data
 base.

 Conclusions
 1. No statistical difference in cadmium
   exposure  rate is demonstrated be-
   tween the non-vegetarians and lacto-
   ovo-vegetarians of this study.
 2. The cadmium exposure rate of the
   small group of pure vegetarians
   studied  is  in the direction anticipated
   (higher than non-vegetarians), but is
   not large enough to achieve statisti-
   cal  significance.
 3. Cadmium  accumulation in the cntial
   organ (kidney) of subjects coming to
   autopsy is well below the range as-
   sociated with renal injury.
 4. Quantitative methods for measuring
   total urine protein are not as satisfac-
   tory for assessing tubular proteinuna
   as those developed  to measure the
   small proteins that normally appear
   in the glomerula filtrate.
 Discussion
  Anticipated  differences  in cadmium
 exposure rates between  vegetarians
 and non-vegetarians are dependent
 upon the replacement of cadmium-rich
 calories from cadmium-accumulating
 foods  by  cadmium-poor calories.  For
 this difference to be apparent, the food
 of the vegetarian  must contain more
 cadmium than  that  of the non-
 vegetarian. The ideal situation for dem-
 onstrating the difference is in the case
 of a single major food source for each
 group. This was the case for the Japa-
 nese  eating rice grown in cadmium-
 contaminated water. But for the situa-
 tion where there are a variety of foods,
 the difference may be masked. The
 non-vegetarian may  be  getting  higher
 than background exposure  by including
 liver,  kidney and shellfish  in this diet.
 The vegetarian who obtains  the major
 portion of his food from the national
 food distribution system averages  his
 exposure by the mixing of foods grown
 in cadmium-poor districts with those of
 contaminated areas.  Another difficulty
 in demonstrating differences is in the
 classification  of the subjects.  In the
 strictest sense there are  no pure vege-
3

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  tarians unless they grow  all  the food
  they eat. Processed foods usually in-
  clude milk  or  egg solids and other
  non-vegetable additives and anyone de-
  pending upon the prepared food indus-
  try is getting more than he  realizes. Self
  assignment  to  pure vegetarian, lacto-
  ovo-vegetarian  or non-vegetarian
  groups may represent the  person's
  ideal for his dietary practice, rather than
  his invariant practice. Of those pure
  vegetarians  in the Adventist Health.
  Study we contacted, most used some
  dairy  products  occasionally.  Even  the
  non-vegetarian group may  be subject to
  cultural influence from its Adventist
  background  to  eat less meat than  the
  general population does.
    The lack of difference  between  the
  non-vegetarian group and the lacto-
  ovo-vegetarian  group in this study is
  probably due to several factors. In spite
  of attempts to control confounding fac-
  tors by matching  subjects  by  age, sex,
  occupational group, etc., there still re-
  main  many  differences (genetic, geo-
  graphic origins, past changes in life-
  style) to mask diet-induced differences.
  Both groups are subject to environmen-
  tal contamination from tobacco smoke
  and other erratic sources. Both replace a
  portion of the higher cadmium content
  plant foods  in their diet by cadmium-
  poor foods—dairy products for the veg-
  etarians and skeletal muscle meat cuts
  for the non-vegetarians. It is unfortu-
  nate that we could not recruit a larger
  group of pure vegetarians to reduce the
  dietary overlap.
    The subjects in the autopsy study are
  not easily categorized into well-defined
  dietary groups. A few were lifelong
  lacto-ovo-vegetarians but many  be-
  came Adventists  later in  life  and had
       made  significant life style and dietary
       changes.  Several had been heavy
       cigarette smokers. Thus, the probable
       rate of exposure had varied significantly
       at different times of their lives. Even so,
       the total renal cortex accumulation re-
       mained below the risk threshold of 200
       micrograms cadmium per gram of wet
       tissue.
        The concentration of cadmium in
       flour used in making  meat analogues is
       in the  range reported by other studies.
       The highest value was from  a sample
       obtained from a partially open flour bin
       in a market where it may have been con-
       taminated by the environment.
        Urine cadmium concentration was at
       the low end of the working curve of the
       instrument. Its measurement was facili-
       tated  by the concentration achieved by
       the extraction step.
      .  Urine protein  methods are tuned to
       measure albumin. In the typical clinical
       setting, the physician is most interested
in proteinuria as an indicator  of
glomerular dysfunction. Since the
major protein appearing in the urine as
a consequence of glomerula injury is
albumin, such methods meet the clini-
cal need. They vary widely in their re-
sponse to other proteins. The smaller
proteins normally pass through the
glomerular basement membrane, but
are removed form the  glomerular fil-
trate by the tubular epithelium. In the
setting of tubular dysfunction or injury
these small proteins replace albumin as
the constituents of greatest clinical in-
terest, but most methods grossly under-
estimate  or fail to detect them. In our
study, this methodological shortcoming
is evidenced by the lack of correlation
between beta-2-microglobulin excre-
tion and total protein excretion. Tubular
proteinuria must be looked for specifi-
cally by urine protein electrophoresis or
by specific assay of one or more of the
small protein species.
         R. E. Willard is with Loma Linda University Medical Center, Loma Linda, CA
           92354.
         Norman Kowal is the EPA Project Officer (see below).
         The complete report, entitled "Assessment of Cadmium Exposure and Toxicity
           Risk in an American Vegetarian Population," {Order No. PB 85-211  340/AS;
           Cost: $10.00, subject to change) will be available only from:
                National Technical Information Service
                5285 Port Royal Road
                Springfield, VA 22161
                 Telephone: 703-487-4650
         The EPA Project Officer can be contacted at:
                Health Effects Research Laboratory
                U.S. Environmental Protection Agency
                Research Triangle Park, NC 27711
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
Official Business
Penalty for Private Use $300
EPA/600/S1-85/009
                                                                                    •frU.S. GOVERNMENT PRINTING OFFICE:1985—559-016/2

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