Environmental Health Perspectives • VOLUME 108 | NUMBER 7 | July 2000 611

Analysis of Hydroxylated Metabolites of PCBs (OH-PCBs) and Other

Chlorinated Phenolic Compounds in Whole Blood from Canadian Inuit

Courtney D. Sandau,1,2 Pierre Ayotte,3 Eric Dewailly,3 Jason Duffe,2 and Ross J. Norstrom1,2

1Centre for Analytical and Environmental Chemistry, Department of Chemistry, Carleton University, Ottawa, Ontario, Canada;

2Environment Canada, Canadian Wildlife Service, National Wildlife Research Centre, Hull, Québec, Canada; 3Unité de recherche en santé

publique, Centre de Recherche du CHUL–Centre Hospitalier Universitaire de Québec, Beauport, Québec, Canada 

Polychlorinated biphenyls (PCBs) have been

extensively studied since their discovery as

environmental pollutants over 30 years ago

(1). Their persistence in biota is well known

but the mechanism of their adverse effects

on biologic systems is still not completely

understood. Some of their toxicity may be

linked to the biotransformation products of

PCBs (2). PCBs are biotransformed by a

diverse enzyme system, the cytochrome

P450 monooxygenases. Most of the known

metabolic pathways involve the initial formation of hydroxylated metabolites. Even

one of the more recalcitrant PCBs, CB153,

is metabolized in vitro and in vivo to a number of phenolic metabolites (3–5). Phenolic

metabolites can be excreted unchanged, as

they were first discovered in the excreta of

Baltic seals and Guillemots (6), or further

conjugated with glucuronic acid or sulfate

(7). The introduction of a hydroxyl group

increases the polarity of the PCB and

facilitates excretion. If the hydroxyl group is

para to the phenyl–phenyl bond and has

adjacent chlorine atoms, the structure resembles the prohormone, thyroxin (T4) (8). This

structural similarity allows hydroxylated

polychlorinated biphenyls (OH-PCBs) to

bind with high affinity to one of the thyroid

hormone transport proteins, transthyretin

(TTR) (9–12). For example, 4-OH-3,3 ́,4,5 ́-

tetrachlorobiphenyl, a metabolite of CB77,

has a binding affinity to TTR 4 times

stronger than that of T4 (9), and can disrupt

thyroid hormone and retinol (vitamin A)

transport (13). This is the presumed mechanism by which OH-PCBs are selectively

retained in plasma and is a possible mechanism of PCB toxicity (14). This type of

interaction is not limited to OH-PCBs.

Pentachlorophenol (PCP) binds with 2

times the affinity of T4 to TTR (15), indicating that other chlorinated phenolic compounds may also be interfering in thyroid

hormone transport.

Some Inuit consume traditional foodstuffs consisting of fatty tissues from sea

mammal species such as ringed seal and beluga (16–18). Ringed seal blubber, beluga skin,

and beluga blubber in northern Canada contain average total PCB concentrations of

1,283, 145, and 5,000 ng/g wet weight,

respectively (19,20), which is much higher

than that in the diet of the general population

(21). Thus, the Inuit population may be

exposed to large doses of PCBs. In one

study, mean PCB blood levels in Inuit were

30 times those of a southern population

(22). An increased PCB body burden could

result in the increased formation of metabolites due to induction of the P450 system.

Increased levels of metabolites may be significant enough to disrupt thyroid hormone

transport. Alterations of thyroid hormone

status have been proposed as a mechanism of

action by which PCBs would induce adverse

neurodevelopmental effects (23). It is therefore of interest to study the concentrations

of OH-PCB metabolites in the Inuit population as a first step to evaluate their possible

implications mediating PCB-induced health

effects. Hydroxylated PCBs have never been

studied in the Arctic environment and little

is known about levels and patterns in

humans. There are only two other published

studies on OH-PCB metabolite concentrations in human blood [from Sweden and

Latvia (14,24)].

Materials and Methods

During fall 1992, 499 Inuit adults living in

Nunavik (Northern Quebec, Canada) participated in the Santé Québec Health Survey.

After they signed an informed consent form,

we drew a 30-mL blood sample by venous

puncture for organochlorine determination.

We randomly selected 30 subsamples of these

499 samples for chlorinated phenolic compound and additional PCB residue analysis.

The Laval University Medical Centre (SainteFoy, Québec) donated a southern Quebec

general population sample of pooled whole

blood for comparison to the Inuit population.

The chosen nomenclature for numbering hydroxylated PCBs or their derivatized

Address correspondence to R. Norstrom, Environment Canada, Canadian Wildlife Service, National

Wildlife Research Centre, 100 Gamelin Boulevard,

Hull, Québec, K1A OH3, Canada. Telephone:

(819) 997-1411 Fax: (819) 953-6612. E-mail:

ross.norstrom@ec.gc.ca

We thank Å. Bergman and E. Klasson-Wehler for

their contributions toward this publication. We

obtained informed consent from all individuals

before their participation in this study. 

The project was funded by the Canadian

Chlorine Coordinating Committee (C4) and the

Canadian Chemical Producers Association. 

Received 20 September 1999; accepted 17

December 1999.

Articles

In this study, we identified the main hydroxylated polychlorinated biphenyls (OH-PCBs) and

other chlorinated phenolic compounds and we determined their relative concentrations in whole

blood from 13 male and 17 female Inuit from northern Quebec, Canada, and from a pooled

whole blood sample from southern Quebec. We also determined concentrations of polychlorinated biphenyls (PCBs). Total OH-PCB concentrations were variable among the Inuit samples,

ranging over 2 orders of magnitude (0.117–11.6 ng/g whole blood wet weight). These concentrations were equal to and up to 70 times those found for the southern Quebec pooled whole blood

sample. Geometric mean concentrations of total OH-PCBs were 1.73 and 1.01 ng/g whole blood

for Inuit men and women, respectively, and 0.161 ng/g whole blood for the southern population

pool. There are limited data available for comparison, but the levels of OH-PCBs in Inuit are

higher than those previously reported in the literature for other populations. There was a significant correlation (p < 0.005) between OH-PCBs and PCBs (r = 0.84) and both correlated significantly (p < 0.005) with age (r = 0.68 and 0.78, respectively). The ratio of OH-PCBs to PCBs was

lower in Inuit (0.11) than in the southern Quebec pool (0.33). There is no apparent explanation

for the difference. There was considerable variability in the congener pattern of the identified

OH-PCBs. The main metabolite, 4-OH-CB109 (4-OH-2,3,3 ́,4 ́,5-pentachlorobiphenyl), constituted 12–62% of the total OH-PCBs in the samples. Pentachlorophenol (PCP) was the dominant phenolic compound in blood, constituting 46% (geometric mean) of the total quantitated

chlorinated phenolic compounds. PCP concentrations in Inuit blood ranged from 0.558 to 7.77

ng/g on a wet weight basis. All but two Inuit samples had lower concentrations than the southern

Quebec pool (6.29 ng/g). The possible role of OH-PCBs in mediating PCB-induced adverse

effects needs to be investigated further. Key words: Canadian Inuit, chlorinated phenolic compounds, hydroxylated metabolites, OH-PCBs, PCBs, pentachlorophenol, polychlorinated

biphenyls. Environ Health Perspect 108:611–616 (2000). [Online 25 May 2000]

http://ehpnet1.niehs.nih.gov/docs/2000/108p611-616sandau/abstract.html
analogs, methoxylated PCBs, is slightly different from general International Union of

Pure and Applied Chemistry (IUPAC)

guidelines (25). The OH- or MeO- groups

are not given numbering priority. Rather, the

chlorine pattern on the biphenyl rings determines the congener number according to the

IUPAC PCB numbering rules (25), with corrections by Guitart et al. (26), and the OHor MeO- groups are numbered thereafter.

This facilitates direct structural comparison

with PCBs because the IUPAC numbering

system is so well established and familiar.

We used the following 13C12-labeled standards [acquired from Wellington Laboratories

(Guelph, Ontario, Canada)] as recovery internal standards for OH-PCB determination:

4 ́-OH-CB120 (4 ́-OH-2,3 ́,4,5,5 ́-pentachlorobiphenyl); 4 ́-OH-CB159 (4 ́-

OH-2,3,3 ́,4,5,5 ́-hexachlorobiphenyl);

4 ́-OH-CB172 (4 ́-OH-2,2 ́,3,3 ́,4,5,5 ́-

heptachlorobiphenyl); and 4-OH-CB187

(4-OH-2,2 ́,3,4 ́,5,5 ́,6-heptachlorobiphenyl). We purchased PCP (13C6) from

Cambridge Isotope Laboratories (Andover,

MA) and used it for PCP quantitation. We

added a synthesized performance standard,

4 ́-Me-4-MeO-2,3,3 ́,5,6-pentachlorobiphenyl, to all phenolic compound fractions before mass spectral analysis. We used

labeled PCBs (13C12-CB28, -52, -118, -153,

-180, and -194) as recovery standards and

13C12-CB138 as the performance standard

for PCB analysis by the external standard

method. The 13C12-PCB standards were purchased from Cambridge Isotope Laboratories.

Whole blood samples (mass range

1.54–5.76 g) were spiked with 13C12 recovery

standards before extraction. The standards

consisted of a PCB mixture (13C12-CB28,

-52, -118, -153, -180, and -194; 10 μL, 2.5

ng/μL), a OH-PCB mixture (13C12-4 ́-OHCB120, 4 ́-OH-CB159, 4 ́-OH-CB172,

and 4-OH-CB187; 20 μL, 100 pg/μL), and

PCP (13C6; 100 μL, 100 pg/μL). The

samples were then extracted using the

Wallenberg plasma extraction method, as

described elsewhere (14,27), with the following modifications. The organic phase from

the potassium hydroxide partitioning, which

contains the neutrals, was reduced in volume

and applied to a Florisil column (8 g, 1.2%

deactivated). The PCBs were collected in

one fraction of 75 mL dichloromethane

(DCM):hexanes (1:1) then reduced in volume and applied to a silica/sulfuric acid (3 g,

22%) column to remove coextracted biogenic components. The PCBs were eluted in

50 μL DCM:hexanes, reduced to final volume (50 μL) by rotoevaporation, and spiked

with performance standard (13C12; CB138,

5 μL, 2.0 ng/μL) for mass spectral analysis.

The partitioned phenolic compounds

were acidified and back extracted with 

hexanes, dried over sodium sulfate, and

derivatized with diazomethane. We generated

diazomethane as needed from nitrosomethylurea precursor, as described elsewhere (28).

The derivatized compounds were then

cleaned up on a silica/sulfuric acid (5 g,

22%) column and eluted with 50 mL

DCM:hexanes. We reduced the samples in

volume by rotoevaporation and brought the

samples down to final volume (25 μL) by a

gentle stream of nitrogen. All samples were

then spiked with a performance standard

(4 ́-Me-4-MeO-2,3,3 ́,5,6-pentachlorobiphenyl, 5 μL, 200 pg/μL) before analysis.

Analyses were completed on a Hewlett

Packard 5890A Series II gas chromatograph

Hewlett Packard, Atlanta, GA) equipped

with an HP 7673A automatic injector and a

Hewlett Packard 5988A mass spectrometer.

We used helium as the carrier gas; we set the

head pressure at 80 kPa. All injections (2 μL)

were made in splitless mode onto a DB-5

column [(5%-phenyl)-methylpolysiloxane,

30 m × 0.25 μm i.d., 0.25-μm film thickness; J&W Scientific, Inc., Folsom, CA).

The injector temperature was set at 250°C.

We performed mass spectrometry with

selected ion monitoring in electron capture

negative ionization (ECNI) mode using

methane (99.99% pure) as the reagent gas

for the chlorinated phenolic fraction. The

reagent gas source pressure was 2.5 × 10-4

torr at the inlet and the source temperature

was 140°C. The GC temperature program

for the derivatized phenolic compounds was

as follows: 80°C for 2 min, ramp 10°C/min

to 250°C, hold for 5 min, then ramp

5°C/min to 300°C. We analyzed the phenolic compound fraction using ECNI because

of the presence of residual biogenic material

that interferes with the analysis in electron

impact (EI) mode. The PCB fraction was free

of biogenic contamination, allowing analysis

of the fraction using selected ion monitoring

in EI mode. The electron energy was 70 eV

and the source temperature was 200°C. The

GC temperature program for the PCB analysis was 100°C for 3 min, 10°C/min to 180°C,

and then 2.5°C/min to 280°C.

Because analysis was carried out on

methylated OH-PCBs, we used a standard

mixture of MeO-PCBs. The MeO-PCB

mixture included 4 ́-MeO-2,3 ́,4,5,5 ́-pentachlorobiphenyl (4 ́-MeO-CB120);

4 ́-MeO-2,3,3 ́,4,5 ́-pentachlorobiphenyl

(4 ́-MeO-CB107); 4-MeO-2,2 ́,3,4 ́,5,6,6 ́-

heptachlorobiphenyl (4-MeO-CB188);

3-MeO-2,2 ́,4,4 ́,5,5 ́-hexachlorobiphenyl

(3-MeO-CB153); 4-MeO-2,2 ́,3,4 ́,5,5 ́-

hexachlorobiphenyl (4-MeO-CB146);

3 ́-MeO-2,2 ́,3,4,4 ́,5 ́-hexachlorobiphenyl

(3 ́-MeO-CB138); 4 ́-MeO-2,2 ́,3,3 ́,4,5 ́-

hexachlorobiphenyl (4 ́-MeO-CB130); 3 ́-

MeO-2,2 ́,3,4 ́,5,5 ́,6-heptachlorobiphenyl

(3 ́-MeO-CB187); 4 ́-MeO-2,2 ́,3,3 ́,4,5 ́,6-

heptachlorobiphenyl (4 ́-MeO-CB175);

4 ́-MeO-2,3,3 ́,4,5,5 ́-hexachlorobiphenyl

(4 ́-MeO-CB159); 3 ́-MeO-2,2 ́,3,4,4 ́,5,5 ́-

heptachlorobiphenyl (3 ́-MeO-CB180);

4 ́-MeO-2,2 ́,3,3 ́,4,5,5 ́-heptachlorobiphenyl (4 ́-MeO-CB172); and 4-MeO2,3,3 ́,4 ́,5,5 ́,6-heptachlorobiphenyl

(4-MeO-CB193). The MeO-PCBs were

supplied by Å. Bergman (Wallenberg Laboratories, Stockholm, Sweden), and synthesized as described elsewhere (29). We used

the MeO-PCBs for quantitation by the

external standard method.

We used a serial dilution of the MeOPCB mixture for quantitation. We generated

response factors relative to the performance

standard (RRFs) for each of the compounds

in the standard mixture. The RRFs were

then used to quantitate all identified and

unidentified OH-PCBs in each sample by

the external standard method, with volume

correction using the performance standard.

Identified compounds were quantitated using

their RRF. Structures were considered confirmed if they had identical spectra and

matching retention times on three GC

columns with varying polarities (DB5,

DB1701, and DB210; J&W Scientific, Inc.).

We characterized unidentified compounds by

full scan mass spectrometry to determine the

chlorination pattern. The average RRFs for

that chlorination pattern were used for quantitation for the unidentified compounds. The

RRFs for compounds with a given chlorination and methoxy group substitution were

comparable. For example, the RRFs for the

para-methoxylated heptachlorobiphenyls were

all within 10% of the average RRF. We determined recoveries using the 13C12 OH-PCB

standards and 4 ́-Me-4-MeO-2,3,3 ́,5,6-pentachlorobiphenyl as the performance standard.

PCP was quantitated by isotope dilution

using the 13C6 internal recovery standard correcting for the 3.4% contribution of native

PCP (m/z = 280 amu) to the main ion of the

13C6 standard (m/z = 286 amu) cluster.

PCBs were quantitated on a congenerspecific basis by the external standard

method using an Aroclor 1242:1254:1260

(1:1:1) secondary quantitation standard solution calibrated against primary standard PCB

congener solutions provided by the National

Research Council of Canada (Marine

Analytical Chemistry Standards Program,

Halifax, Nova Scotia, Canada). We determined concentrations of congeners for which

standard solutions were not available in the

Aroclor mixture by GC using flame ionization detection (30). We purchased Aroclor

from Monsanto (St. Louis, MO).

PCBs were previously determined in

these samples by the Québec Toxicology

Centre (Sainte-Foy, Québec, Canada) using

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612 VOLUME 108 | NUMBER 7 | July 2000 • Environmental Health Perspectives
plasma as the tissue for analysis. Because of

Aroclor 1254 contamination in one of our

rotoevaporators, 15 of the 30 whole blood

samples were irreclaimable for PCB quantitation. We compared the 15 whole blood

samples not contaminated with Aroclor

1254 to the previous plasma analyses.

Concentrations of all previously quantitated

congeners (CB28, CB52, CB99, CB101,

CB105, CB118, CB128, CB138, CB153,

CB156, CB170, CB180, CB183, and

CB187) and the sum of congeners in plasma

were highly correlated (r > 0.97) to the

whole blood concentrations using leastsquares regression analysis. The regression

analysis for CB153 and total PCBs are given

as examples in Figure 1. Thus, the previous

PCB plasma concentrations for the 15 samples not quantitated in our lab were adjusted

by applying a correction factor from the

regression analysis. We used the adjusted

PCB concentrations and the values determined in the 15 whole blood samples in the

present study for comparison to OH-PCB

concentrations and for subsequent statistical

analysis. Aroclor contamination had no

effect on the quantitation of OH-PCBs in

the contaminated samples because PCBs

elute earlier and have no fragment ions that

interfere with the detection of OH-PCBs.

All solvents were residue analysis grade

and were purchased from EM Science

(Gibbstown, NJ). Florisil (Pesticide Analysis

Residue grade, 60–100 mesh) was purchased

from BDH, Inc. (Toronto, Ontario, Canada). Merck Silica gel (Grade 60, 70–230

mesh, 60Å) was purchased from Aldrich

Chemical Company, Inc. (Milwaukee, WI).

We purchased trace-metal-grade sulfuric acid

from Fisher Scientific (Pittsburgh, PA).

Results

Whole blood recoveries of OH-PCBs were

variable, ranging from 50 to 105%. The

mean recoveries for 13C12- 4 ́-OH-CB120,

-4 ́-OH-CB159, -4 ́-OH-CB172 and -4-

OH-CB187 were 82, 69, 72, and 76%,

respectively. This was the first study to use

13C12-labeled OH-PCBs for accurate

OH-PCB quantitation. PCB recoveries had a

slightly lower mean recovery of 60%. All PCB

and OH-PCB concentrations were consequently recovery corrected.

An example chlorinated phenolic ECNIMS full scan chromatogram is given in

Figure 2. The major peaks are identified

when known. The OH-PCB fraction in

Inuit whole blood contained > 30 congeners,

of which 11 were positively identified with

authentic standards. Positive identification is

based on identical mass spectra using full

scan ECNI mass spectrometry and matching

retention times using three GC columns

with varying polarities (results not shown).

All identified congeners are listed in Table 1.

The congeners constituted between 59 and

81% of the total OH-PCBs in the samples

(mean = 70%). 

We did not determine the lipid content of

the whole blood samples. Therefore, all concentrations hereafter are expressed on a whole

blood wet weight basis. The chemical residue

data were not normally distributed. Logtransformed data approached normal distribution; therefore, log-transformed data were

used in all statistical analyses and geometric

mean values were used unless otherwise stated. The geometric mean concentrations in

men and women of all identified congeners

and their range of values are listed in Table 1.

Women consistently had lower geometric

mean concentrations of all the chlorinated

phenolic compounds quantitated, but the

difference was statistically significant between

men and women using Student’s t-test 

only for PCP.

The concentrations in the pooled 

sample are equivalent to arithmetic mean

concentrations. The arithmetic mean of the

total OH-PCBs in Inuit whole blood was

1.89 times greater than the geometric mean.

Therefore, the difference in geometric mean

values between populations is probably an

overestimate of the geometric mean and the

differences in concentrations are even greater

than indicated in Table 1.

PCP was the main chlorinated phenolic

compound in the Inuit whole blood. It contributed between 14 and 89% of the total

chlorinated phenolic compounds quantitated. The concentration of PCP ranged from

0.558 to 7.77 ng/g with a geometric mean of

2.02 ng/g wet weight. The pooled whole

blood sample from the southern population

had higher PCP levels than all but two Inuit

samples analyzed, with a concentration of

6.29 ng/g. PCP composed 97% of the total

quantitated chlorinated phenolic compounds in the southern population.

The major OH-PCB congeners varied

considerably among individuals. The mean

ratios of the five main congeners to total

OH-PCBs are shown in Figure 3, with bars

representing the range. The main congener

identified in Inuit whole blood (21 of 30

samples) was 4-OH-CB109, with a geometric

mean concentration of 0.266 ng/g and a

range of 0.015–2.55 ng/g. This congener is

probably a mixture of two congeners, 4-OHCB109 (4-OH-2,3,3 ́,4 ́,5-pentachlorobiphenyl) and 4 ́-OH-CB107, because they

coelute and because separation was not

achieved with the DB-5 column used in this

study. This peak was quantitated using only

4 ́-OH-CB107 as the quantitation standard

because 4-OH-CB109 was not commercially

available. The peak is assumed to be predominantly 4-OH-CB109 because it is the dominating compound of the mixture in human

plasma at a 5:1 excess (14,31). However, considering pattern variability this would need to

be confirmed for each sample in the future.

Articles • Hydroxylated metabolites in Inuit whole blood

Environmental Health Perspectives • VOLUME 108 | NUMBER 7 | July 2000 613

Figure 1. The regression analysis of (A) CB153 and (B) total PCBs, showing that the concentrations previously determined in plasma are significantly correlated to those in whole blood determined in this study.

The correction factor was developed by regression of the whole blood concentration versus the plasma

concentration from the previous analyses. The equation for the least-squared regression can then be

used to adjust the plasma values for the samples into whole blood equivalents. This was done for each

congener independently and for the sum of all quantitated PCBs. (A) r = 0.97, p < 0.005; y = 0.76x - 0.23. (B)

r = 0.98, p < 0.005; y = 0.74x + 5.7. 

16

8

0

0 10 20 0 40 80

A

Whole blood (ng/g)Whole blood (ng/g)

Plasma (ng/mL) Plasma (ng/mL)

70

30

0

B

Figure 2. GC-MS (ECNI) SIM chromatogram of

the OH-PCBs and other chlorinated phenolic

compounds from Inuit whole blood analysis. PCP

was truncated to fit on the scale shown. The

chromatogram can only be used as a qualitative

graph because each compound fragments differently and the response varies for compounds

with different chlorination patterns. For this sample, only two internal standards (IS) and the

performance standard (PS) were added for illustrative purposes. Abundance 0 12 14 16 18 20 22 24 26 28

PCP

UCl6

IS 4-OH-CB120 IS

4’-OH-CB159

4-OH-CB109

3-OH-CB153/4-OH-CB146

4-OH-CB187

PS

3’-OH-CB180

4’-OH-CB172

4-OH-CB193

C18

C19

di-OH-C18

Retention time (min)
In eight of the samples, 4-OH-CB187

was the dominating congener. It ranged

from 0.022 to 2.26 ng/g with a geometric

mean of 0.202 ng/g for all samples. This was

the main metabolite in plasma determined

in previous studies (14,32,33). The main

metabolite in the remaining sample was 4-

OH-CB146. The geometric mean concentration for 4-OH-CB146 in all samples was

0.149 ng/g and ranged from 0.010 to 1.88

ng/g. The metabolite may be formed from

CB146 via a direct oxygen insertion or from

CB153 or CB138 via a National Institutes

of Health (NIH) shift of Cl in the hydroxylation step. If a metabolite is formed by a

direct oxygen insertion, it would retain the

precursor PCB number. If the metabolite is

produced via an NIH shift of Cl, the

metabolite PCB number will be different

from that of the parent PCB.

Total quantitated OH-PCBs ranged

from 0.117 to 11.6 ng/g with a geometric

mean value of 1.27 ng/g. Total OH-PCBs in

individuals were equal to and up to 70 times

that of the southern Quebec pooled sample

(0.161 ng/g). Geometric mean CB153 and

geometric mean total PCB concentrations in

combined Inuit samples were 2.37 ng/g

(range 0.263–13.9 ng/g) and 15.2 ng/g

(range 1.19–65.9 ng/g), respectively.

Concentrations in the southern Quebec

pooled sample were 0.074 ng/g for CB153

and 0.488 ng/g for total PCBs. The mean

ratio of total OH-PCBs to total PCBs was

0.11 (range 0.02–0.45) for combined Inuit

and 0.33 for the southern Quebec pooled

sample, demonstrating the importance of

OH-PCBs in the total PCB-related compounds in blood. The concentration of the

five main OH-PCB metabolites relative to

total OH-PCBs and CB153 for both Inuit

and the southern Quebec pooled sample are

shown graphically in Figure 3.

Plots of log-normalized concentrations

versus age are shown in Figure 4. Both PCBs

(r = 0.78) and OH-PCBs (r = 0.68) were significantly (p < 0.005) correlated with age. As

seen by the similar slopes, total OH-PCBs

and total PCBs were also significantly correlated (r = 0.84, p < 0.005). 

Discussion

The only human blood analyzed previously

for OH-PCBs and other chlorinated phenolic compounds was plasma from the Swedish

population (14,31). PCP was described as

the dominant compound in these studies,

but levels were not given. The geometric

mean PCP concentration in the Inuit samples was 2.02 ng/g, approximately 3 times

lower than in the southern Quebec pooled

sample. Geyer et al. (34) measured PCP in

two different general populations in

Germany and found average concentrations

of approximately 20 ng/g wet weight. The

study did not indicate if the samples analyzed were whole blood or plasma, so the

comparison of levels with the present results

may be incorrect. However, a recent study

documented levels of PCP in men from

Sweden and Latvia between 170 and 1,800

ng/g on a plasma lipid weight basis (24).

When the Swedish data are approximated to

plasma equivalents, these levels are the same

order of magnitude and have a similar range

as those for the Inuit and the southern

Quebec pooled whole blood samples. The

plasma equivalent was estimated by assuming

that whole blood is approximately half plasma

by weight and that the average plasma lipid

levels in the population are approximately 1%.

Although the concentration of PCP is

lower in Inuit than that measured in the

southern Quebec pooled whole blood sample, it may still play a role in the disruption

of thyroid hormone transport. PCP binds to

TTR with twice the affinity of the native

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614 VOLUME 108 | NUMBER 7 | July 2000 • Environmental Health Perspectives

Table 1. Concentration of PCP and hydroxylated metabolites (ng/g whole blood wet weight × 103) in Inuit

whole blood.a

Males (n = 13) Females (n = 17) Southern

Range Range population

GM Min Max GM Min Max pool

Age (years) 38 18 66 38 18 72 –

PCP 2,740 1,350 7,770 1,590 558 7,510 6,290

4 ́-OH-CB120 12 3 67 7 < 1 39 3

4-OH-CB109b 314 39 2,550 234 15 1,470 25

3-OH-CB153b 85 5 537 49 4 830 2

4-OH-CB146b 219 10 1,750 111 10 1,880 8

3 ́-OH-CB138 37 3 225 20 2 401 2

4 ́-OH-CB130 15 1 506 4 < 1 47 < 1

3 ́-OH-CB187 17 1 213 11 < 1 167 < 1

4-OH-CB187b 293 22 1,840 152 27 2,260 31

3 ́-OH-CB180 17 2 134 8 1 239 < 1

4 ́-OH-CB172b 74 5 443 38 4 740 11

4-OH-CB193 41 3 659 26 2 95 18

Sum congenersb 1,040 88 6,740 614 59 7,070 76

Sum identified 1,210 119 7,520 712 87 8,060 82

Sum all OH-PCBs 1,730 162 10,100 1,010 117 11,600 161

Sum PCBs 12,900 2,070 65,900 7,940 1,190 38,100 488

CB-153 3,120 460 13,900 1,960 263 9,510 74

Abbreviations: GM, geometric mean; max, maximum; min, minimum. 

aTotal OH-PCBs, sum five main congeners, and sum identified congeners are given for comparison with previous studies.

bSum five main congeners: 4-OH-CB109 + 3-OH-CB153 + 4-OH-CB146 + 4-OH-CB187 + 4 ́-OH-CB172. 

Figure 3. Ratio of the five main metabolites to total OH-PCBs and to CB153. Mean ratios are given for combined male and female Inuit samples, and error bars represent the range. 

0.8

0.6

0.4

0.2

0

Ratio to total OH-PCBs

4-OH-CB1093-OH-CB1534-OH-CB1464-OH-CB187

4’-OH-CB172

4-OH-CB1093-OH-CB1534-OH-CB1464-OH-CB187

4’-OH-CB172

0.8

0.6

0.4

0.2

0

Ratio to CB153

A B Inuit

Southern pool

Figure 4. Log-normalized concentrations versus

age (in years) for total PCBs and total OH-PCBs.

PCBs, Y = 0.022x + 3.10; r = 0.78; p < 0.005.

OH-PCBs, Y = 0.023x + 2.17; r = 0.68; p < 0.005. 

100

10

1

0.110 20 30 40 50 60 70 80

Age (years)

Concentration (ng/g)

PCBs

OH-PCBs
hormone (15) and is responsible for

14–89% of the total phenolic compounds

quantitated. Geyer et al. (34) calculated the

half-life of PCP as approximately 19 days

and concluded that the German cohort’s

calculated intake of PCP exceeded the elimination rate and approximated a bioconcentration factor of 1.4 for blood. This is likely

due to binding with TTR and explains the

high levels of PCP found in blood. Considering the significance of PCP in the chlorinated phenolic fraction, further investigation

of PCP and its possible effects on thyroid

hormone transport is required, especially for

southern populations, where levels of PCP

far exceed OH-PCBs and other chlorinated

phenolic compounds.

Another major compound in the Inuit

whole blood chromatogram is an unidentified chlorinated phenolic compound that

elutes just before the OH-PCBs. The major

ion cluster for this compound, using the

given mass spectrometry conditions, has an

isotope pattern indicative of a hexachlorinated compound. Therefore, the levels were

approximated using the Cl6 response factor

from the OH-PCB standard mixture. Based

on approximate concentrations, the compound accounts for 1–17% of total chlorinated phenolic compounds in the Inuit.

Levels occasionally exceeded those of the

main OH-PCBs. Further work on the identification and accurate quantitation of this

unknown compound is currently underway.

The chromatogram of the phenolic compound fraction consists of > 30 peaks that

were identified as hydroxylated metabolites

of PCBs based on their mass spectra. These

represent 20% of the total chlorinated phenolic compounds, on average, in Inuit whole

blood (range 2–56%). OH-PCBs constitute

a significantly more important proportion of

the total chlorinated phenolic compounds in

Inuit as compared to the general population

pooled sample, in which OH-PCBs represent only 1.2% of the estimated total.

The mean concentration of the five main

OH-PCBs (1.47 ng/g) in Inuit samples is

slightly higher than that of a Swedish general

population cohort (1.0 ng/g) (31). The

Swedish data have been adjusted to whole

blood wet weight equivalents. It was expected that levels of OH-PCBs in Inuit would be

higher than those found because the mean

PCB levels in plasma were 3 times that of

this particular Swedish population, and an

average of 30 times (range 2.5–133) that of

the southern Quebec pooled sample. The

range of the sum of these five congeners in

the Swedish study (0.35–1.65 ng/g) is small

as compared to concentrations in the Inuit

population, which range from 0.059 to 7.07

ng/g plasma wet weight levels of OH-PCBs.

The higher levels of OH-PCBs are likely due

to the higher levels of parent PCBs.

OH-PCBs were significantly correlated with

PCBs (r = 0.84, p < 0.005), as expected. 

The strong linear relationship observed

between concentrations of PCBs and their

metabolites suggests that no changes in

enzyme activity affecting the rate of formation or saturation of OH-PCBs binding to

plasma proteins are taking place for this

range of PCB exposure. The concentration

of total quantitated chlorinated phenolic

compounds was < 2% of the theoretical

molar circulating concentration of TTR in

an average human (35). Thus, it is not likely

that the concentration of total chlorinated

phenolic compounds reached the carrying

capacity of the blood, assuming that Inuit

have similar TTR concentrations as the published values from other populations (35). 

Women had lower mean concentrations

of all phenolic compounds quantitated than

men. The generally lower levels in women

may result from the loss of OH-PCBs and

PCBs through lactation because both have

been identified in milk (36–38). Both

OH-PCBs and PCBs were significantly correlated with age (r = 0.68 and 0.78, respectively; p < 0.05). The increase of both PCB

and OH-PCB concentrations with age may

be due to the increased exposure with age

(elders’ preference for traditional foods), a

slow excretion rate of PCBs that prevents

steady state from being achieved, or both. A

positive association between age and PCB

levels in breast milk was reported previously

in southern Quebec women (39) and in

Inuit adults from Nunavik (22). 

The concentration of omega-3 fatty acids

in plasma phospholipids is a good indicator

of fish intake (40). In the course of the Santé

Québec Health Survey, polyunsaturated

fatty acid analysis was performed on plasma

samples obtained from 492 Inuit adults.

There was a strong association between

omega-3 fatty acid content in plasma phospholipids and the age of the participants,

with mean (geometric) concentrations of

6.4% [95% confidence interval (CI),

5.9–6.9]; 8.7% (CI, 8.2–9.3); and 12.2%

(11.5–13.0) for the 18–24 year, 25–44 year,

and 45–74 year age groups, respectively

(Fisher exact test: p < 0.0001) (41). Thus, an

increase in the intake of traditional foods

with age is indicated, and is probably partly

responsible for the increase in PCBs with

age. However, indications of increasing concentration of PCBs with age in other populations suggest that slow excretion of PCBs is

also important. 

The main OH-PCBs identified in previous studies were also the dominating congeners in the Inuit population (Figure 2).

The major metabolite in 21 of the 30 samples analyzed was 4-OH-CB109. This was

not the major metabolite found in the study

by Bergman et al. (14), but it was later identified by Klasson-Wehler et al. (31) as the

dominant OH-PCB in another study of

human plasma. The probable mechanism of

formation of this metabolite is direct oxygen

insertion into CB-109 or an NIH shift of Cl

through an arene oxide intermediate of CB118 or CB105. CB118 and CB105 are

major congeners of Aroclor mixtures,

whereas CB-109 represents only a fraction

of a percent of the Aroclor mixture; therefore, the NIH shift mechanism is more likely, and occurs in the mink and the mouse

(42). The majority of metabolites were

highly correlated to all PCB congeners,

making it impossible to determine the most

probable precursors by correlation analysis.

For example, 4-OH-CB109 was significantly correlated to both CB105 and CB118

(r = 0.67 and 0.72, p < 0.005). The correlation coefficients for 4-OH-CB109 and

unrelated PCBs, such as CB153 and

CB187, were equally high (0.77 and 0.71,

p < 0.005). This was true for the majority of

the identified metabolites and the major

PCBs in the Inuit whole blood samples.

The main metabolite in most of the

remaining samples was 4-OH-CB187,

which was previously identified as the major

metabolite in a Swedish population (14).

This compound was also identified as the

main metabolite for the southern Quebec

pooled sample and in other species including

polar bears (33) and albatrosses (32). This

metabolite is most likely formed from the

metabolism of CB183 and/or CB187, which

represent 2 and 5%, respectively, of the

Aroclor 1254 mixture (43). 

The OH-PCB pattern present in the

chlorinated phenolic compound fraction in

plasma is complex and it is difficult to generalize patterns in humans from such a small

data set. The ratio of 4-OH-CB109 to total

OH-PCBs ranged from 0.12 to 0.62 (mean =

0.34) (Figure 3). The remaining congeners in

the Inuit all composed similar proportions of

total OH-PCBs as compared to the southern

population. The ratio of the five main congeners to CB153 in the southern pooled samples was within the range of those determined

for the Inuit samples, except for 4-OHCB187, where the southern pooled sample

was higher than all of the Inuit samples.

A number of factors, which include

exposure to different proportions of precursor PCBs, alteration of metabolism rates by

induction of hepatic enzymes, inhibition of

the metabolizing enzymes and the proteinbinding specificity of the plasma, may all

influence the retention of hydroxylated

metabolites. Genetic diversity among individuals and populations may also influence

the metabolism rates and binding specificity. 

Articles • Hydroxylated metabolites in Inuit whole blood

Environmental Health Perspectives • VOLUME 108 | NUMBER 7 | July 2000 615
To evaluate the toxicologic significance

of the phenolic fraction, the main compounds must be identified, accurately quantitated, and toxicologic studies must be

undertaken to evaluate the effects of these

compounds on thyroid hormone and retinol

homeostasis and binding at receptor sites.

PCP remains the dominating phenolic compound in Inuit whole blood, even more so

for the southern Quebec pooled sample.

Concentrations of PCP in most of the samples far exceeded the main OH-PCBs. Other

compounds, such as the unknown chlorinated compound, will be researched further to

elucidate structure and possible roles in disruption of thyroid hormone transport. 

In conclusion, total OH-PCB concentrations were 11 and 33% of total PCB concentrations in Inuit and a southern Quebec

pooled sample of whole blood, respectively.

Both total PCB and total OH-PCB geometric mean concentrations were higher for

Inuit than for the southern Quebec pooled

sample and the current literature values (31).

Increased concentrations resulted in increased

OH-PCB levels in Inuit blood, but the ratio

of metabolites to PCBs was not as high as that

found for the southern Quebec pooled sample. Unlike PCBs, the pattern of OH-PCBs

was not consistent among individuals. Three

different congeners alternated as the dominant metabolite of PCBs in the 30 samples

analyzed. A larger study would help elucidate the reasons for the pattern variability,

the lower ratio of OH-PCBs to PCBs in

Inuit, and the possible differences between

the sexes. PCP was the dominant chlorinated phenolic compound in the majority of

the samples and must be included in future

studies that may be evaluating the effects of

phenolic compounds on circulating levels of

thyroid hormones and vitamin A.

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Articles • Sandau et al.

616 VOLUME 108 | NUMBER 7 | July 2000 • Environmental Health Perspectives