measurement of total nickel in body
TRANSCRIPT
Pure & Appl. Chem., Vol. 66, No. 2, pp. 357-372, 1994. Printed in Great Britain. @ 1994 IUPAC
INTERNATIONAL UNION OF PURE AND APPLIED CHEMISTRY
CLINICAL CHEMISTRY DIVISION COMMISSION ON TOXICOLOGY *
Working Party on Nickel Reference Levels?
MEASUREMENT OF TOTAL NICKEL IN BODY FLUIDS
ELECTROTHERMAL ATOMIC ABSORPTION METHODS AND SOURCES OF PREANALYTICAL VARIATION
(Technical Report)
Prepared for publication by DOUGLAS M. TEMPLETON
Department of Clinical Biochemistry, University of Toronto, 100 College St., Toronto, Canada M5G 1L5
*Membership of the Commission during the period (1989-93) when this report was prepared was as follows: Chairman: 1989-91 P. Grandjean (Denmark); 1991-93 Rita Cornelis (Belgium); Secretary: 1989-91 R. Cornelis (Belgium); 1991-93 B. Henizow (Germany); Titular Members: S . S . Brown (1989-91; UK); J. H. Duffus (1991-93; UK); R. F. M. Herber (1989-93; Netherlands); M. Jakubowski (1989-93; Poland); Jytte Molin Christensen (1991-93; Denmark); E. Nieboer (1989-91; Canada); D. M. Templeton (1991-93; Canada); C. Veillon (1989-91; USA); Associate Members: A. Cavalleri (1989-93; Italy); Sheila Dawling (1991-93; UK); AndrCe Lamberty (1989-93; Belgium); S. RendiC (1989-91; Yugoslavia); D. Rutherford (1991-93; Australia); Y. Thomassen (1991-93; Norway); Marie Vahter (1991-93; Sweden); C. Veillon (1991-93; USA); National Representatives: 0. August0 (1991-93; Brazil); I. DCsi (1989-93; Hungary); P. K. Ray (1989-93; India); W. King (1991-93; Ireland); W. A. Temple (1989-93; New Zealand); M. Repetto Jimenez (1989-93; Spain); Zeliha Imre (1989-93; Turkey); Repre- sentative of IUPHAR Section on Toxicology: J. G. Salway (1989-91; USA); Representative of ZUTOX P. Grandjean (1989-93; Denmark).
?Membership of the Working Party: Chairman: E. Nieboer (Canada); Members: R. Cornelis (Belgium); D. Dewally (France); F. W. Sunderman, Jr. (USA); D. M. Templeton (Canada); 0. Vesterberg (Sweden).
Republication of this report is permitted without the need for formal IUPAC permission on condition that an acknowledgement, with full reference together with IUPAC copyright symbol (0 1994 IUPAC), is printed. Publication of a translation into another language is subject to the additional condition of prior approval from the relevant IUPAC National Adhering Organization.
Measurement of total nickel in body fluids: Electrothermal atomic absorption methods and sources of preanalytic variation (Technical Report)
Synopsis
Before attempting to establish reference concentrations of Ni in blood serum or plasma, a laboratory must be working comfortably at the 2 nmol/L level. To achieve this, scrupulous acid-leaching of all materials that come into contact with the sample is essential, and blood collection with non-metalic systems i s advisable. A particle-free environment (clean-air laboratory) seems necessary. Values of Ni in serum above 1.7 to 5.1 nmol/L are suspect; values in the urine of people without occupational or other unusual environmental exposures are an order of magnitude higher. For reported concentrations to be meaningful, the reference population must be thoroughly described in terms of age, sex, place of residence, occupation, diet, alcohol and tobacco use, medications and medical history, as all such factors may influence the actual concentrations of Ni. Sampling procedures must be standardized (for instance for time of day, season, posture of the person, etc.) and reported. Storage, if necessary, should minimize opportunities for sample adulteration, and these must be evaluated empirically. Preparation of samples should also minimize processing; acidification or protein precipitation with ultrapure nitric acid are adequate for clear body fluids. Analysis by ET-AAS with Zeeman correction is the most useful approach currently available.
1. INTRODUCTION
Exposure to Ni is of occupational significance worldwide, occurring not only in those involved in Ni mining, smelting and refining, but also in such diverse settings as the petroleum and electronics industries. Nickel is ubiquitous in our environment; indeed, exposure of the general population through jewelry, coinage and dental alloys is well documented as a frequent cause of contact dermatitis [I, 21, and oral exposure can be greatly increased by leaching of Ni from plumbing fixtures and kitchen utensils. The International Agency for Research on Cancer (IARC) has recently concluded that “There is sufficient evidence in humans for the carcinogenicity of nickel sulfate. ... Nickel compounds are carcinogenic to humans (Group 1) [whereas] Metallic nickel is possibly carcinogenic to humans (Group 2B)” [ 3 1 . Nevertheless, exposure from natural sources is unavoidable.
There is also some evidence that Ni may be essential for human beings, as it is for numerous species of plants and lower animals t 4 1 . Therefore it is of the utmost importance to measure concentrations of Ni in human body fluids, both in order to understand the natural biology of this element and as a reference point for monitoring undue exposures. It is a goal of the IUPAC Commission on Toxicology to recommend reference substance concentrations for Ni in human blood serum or plasma, whole blood, and urine. At present, the accuracy of all published values remains in some doubt, and all but a few must clearly be rejected. The aims
358
Measurement of total nickel in body fluids 359
of t h i s report a r e :
i) t o review t h e recent l i t e r a t u r e i n order t o determine whether an approximate range of N i concent ra t ions i n body f l u i d s can be recommended a t t h i s time;
ii) t o suggest a l e v e l of accuracy t h a t any laboratory must achieve before it can se r ious ly con t r ibu te t o t h e e f f o r t of e s t a b l i s h i n g reference ranges f o r N i i n serum o r plasma;
iii) t o recommend condi t ions of sampling and s to rage t h a t a r e necessary before any ana ly t i ca l method with adequate s e n s i t i v i t y can be used t o e s t a b l i s h meaningful values i n a s p e c i f i e d re ference population; and
iv ) t o descr ibe current methods of measuring concentrations of N i b y e lectrothermal atomic absorption spectrometry (ET-AAS) . A companion document from t h i s Commission w i l l consider sampling c r i t e r i a f o r t r a c e elements i n general . T h i s repor t w i l l dea l with sampling c o n d i t i o n s s p e c i f i c f o r N i . Because s t a t i s t i c a l cons idera t ions f o r sampling a r e not s p e c i f i c f o r N i they a r e not d i scussed h e r e . Some gene ra l a s p e c t s of sample s t o r a g e a r e discussed, again with reference t o N i . Concluding t h a t ET-AAS i s the bes t candidate reference method f o r N i t w e a l s o review c u r r e n t methods of sample preparat ion f o r ana lys i s of b io log ica l f l u i d s f o r N i by t h i s technique.
Use of substance concent ra t ions f o r r epor t ing N i should be encouraged. Nevertheless, w i t h r a r e exceptions, ana lys t s continue t o report mass concentrations of N i i n body f l u i d s i n pg/L. Here we w i l l r e t a i n t h i s l e s s preferable uni t when quoting au thors ' o r i g i n a l data, and include t h e conversion t o molar u n i t s i n parentheses based on an atomic mass of N i of 58.69. A s o l u t i o n w i t h a mass concentrat ion of N i of 1 . 0 pg/L has a substance concentrat ion of 1 7 . 0 nmol/L.
2.
Over t h e pas t decade, i n common w i t h a number of o the r t r a c e elements, t h e t r e n d has been a progress ive lowering of repor ted concentrations of N i . T h u s , the chronology revea ls new record lows f o r t h e serum/plasma value i n pg/L (nmol/L) being set a s follows (mean f s . d . [year] - see Table 1) : 1 .3 f 0.37 ( 2 2 f 6) [1983], 0 . 4 6 f 0 . 2 6 (7 .8 f 4 . 4 ) [1984], 0 . 1 4 f 0 . 0 9 ( 2 . 4 f 1.5) [1989]. W e can conclude a t t h e ou t se t t h a t t he usual range of N i concentrat ion i n serum i s l e s s than 2 0 nmol/L i n heal thy people with no h i s t o r y of excess ive exposure, and t h a t e a r l i e r h i g h e r va lues were a consequence of sample contaminat ion , i nadequa te a n a l y t i c a l s e n s i t i v i t y , o r both. So, only the l i terature from 1983 onward need be ser ious ly considered. We cannot now s t a t e a value of N i i n body f l u i d s with much c e r t a i n t y . Examination of Table 1 suggests t h a t t h e l e v e l of s e r u m N i i s i n t h e range of 1 . 7 t o 3 .4 nmol/L. Concentrations of N i i n u r ine have been measured c o n s i s t e n t l y a t more than 17 nmol/L and therefore a r e probably an order of magnitude higher than i n serum. Intermediate values can be expected i n blood. Before a l a b o r a t o r y can c o n t r i b u t e t o t h e goa l of measuring concen t r a t ions of N i i n blood serum c o l l e c t e d from a chosen re ference populat ion, i t mus t work a t t h e u l t r a t r a c e l e v e l of 2 nmol/L and below. The accurate measurement of N i i n blood and ur ine should present less d i f f i c u l t y f o r ana lys t s who s u c c e s s f u l l y meet t he challenge of serum ana lys i s . T h i s repor t considers approaches t o sample handling t h a t w i l l help reduce the measured concentration i n a zero reference (method blank) f o r N i t o one where i t s accurate measurement i n serum i s f eas ib l e . I t a l so reviews f ac to r s t h a t m u s t be taken i n t o considerat ion when a reference population i s chosen fo r determining a reference value fo r N i .
Ni CONCENTRATIONS IN BODY FLUIDS
Tabl
e 1.
S
elec
ted
stud
ies
of n
icke
l con
cent
ratio
ns in
bod
y flu
ids
w
Q)
0
NS
+ +
+
+ (ne
edle
NS) NS
NS
Examined m
ater
ials
& so
luti
m
forc
onta
min
atio
n
Ni-DMG extraction in
MB
K
r51
(S)
95 f
20
10
(P) 2
2 f
6.3
(B) 4
1 f
8.5
20 (H
x, a
lcoh
ol,
Tob..
Rx.
161
19.4
f 5.4
O
ccup
.)
NS
+ +
- (3
A
mm
onia
buffi
x, D
MG
(B
) 24.0
f 6
.3 9
0 39
.1 f9
.9
9 (S
aliv
a) 42
.7 f
19.0
5
m
39 (S).
30 (B
) W. To
b., O
ccup
.) (S) 7
.8 f
4.4
(B)
21.4
f 5.
6 t81
+
+ +
- (?I
Dep
rot&
ized
sam
ples
Th
orou
gh do
cum
enta
tion o
f ap
para
tus &
solutions
Assessed c
onfo
undi
ng fa
ctor
s - ti
me o
f ser
um se
para
tiop.
sex. ex
erci
se, p
ost-p
rand
~al
& d
id
fluct
uatio
n
undi
gest
edse
nrm
mix
edw
ith
coat
amin
atio
n in
sam
plin
g devices.
fito
n X
-100
, AS
S~
SS
C~
cont
aine
rs. SS n
eedl
es
+ + PI
(S) 4
.8 f
4.1
(B)
5.8 f
4.8
t N
S +
(S) (
med
ian 1
7)
(40
- 51)
+(
SSne
edle
) +
+ +
0
z d x 0 E 0 <
18
10
10
6 30 (f
astin
g)
20
NS
NS
+ +
+ +
-@ro
nase
) -
+ +
+ -(s
onica
tion)
-
NS
+ +
+ +
NS
NS
NS
+(SS
need
le
+ +
+ vs
. teflon
cann
ula)
+ + (
by re
f.)
+ (by
ref.)
+
(by
ref.)
- c
?)
(S) 1
28 f
53
(S) 4
.25
f 1.7
0 14
f 8.5
Serum
dilu
ted with H
N03
/Tri
ton
X-1
00 fo
r dire
ct an
alys
is
(S) 1
1 f 6.
0
(S) 9
5 f
14
(S) 7
(3 - 9
) D
epro
tein
ized
senu
n; c
ompa
red
nd
e vs
cann
ulae
, ope
n vs
. clo
sed
COUe
ctioa
Deproturuzcdsenun
..
43 (Hospital
wo*
m)
(S)3
f3
(v) 2
5 f
25 m
ol
(S) 2
.4 f
1.5
(mea
n f 2
s.d)
lg -
Cle
an m
om. S
aum
:HN
03/T
riton
Mat
aiab
cm
taxk
g sa
mpl
e acid
le
ache
d, N
eedl
e blo
od ri
nsed
contamination c
ontro
l discussed
X-1
00 3:
1; m
ultip
le in
jcctim
38
(Phy
sica
l &lab
work;
fasti
ng)
+(SS
necd
le)
+ +
+ +
Tabl
e 7
(co
ntd
.)
+ i
+ (S) 2
0 f
1.3
385(
Dem
ogra
phie
i
Mdt
iele
mm
stud
s sp
ecifi
cs
(B) 3
9 f
2.7
36
excl
usio
n fo
r illn
ess.
for N
i not
giv
en.
(v)
15 f 1
.9 87
8 Rx. T
ob..
phys
iol.
stre
ss)
[ml
(S) 6
32 f 1
39
20
NS
+ i
Prea
ncen
mtio
n by
che
latio
n
Not
es to
Tab
le 1
ques
tionn
aire
; r1
91
01
Met
hods
N
otes
RC
fCXXN
X Q
uality
R
efer
ence
m
ater
ials
Ran
Pe
151
GF-AAS
Stan
dard
addi
tions
161
GF-
AA
S he
cisi
on &
reco
very
repo
rted
DP-
volta
mm
. R
eage
nt b
lank
s rep
olte
d St
anda
rd ad
ditio
ns.
+ + + + i
i
- (Liv
er)
+ + - (3
ZGF-
AA
S
Z-G
F-A
AS
GF-
AA
S
GF-AAS
Z-GF-AAS
Z-GF-AAS
Flam
e AAS
GF-AAS
ZGF-
AAS
ZGF-
AA
S
Z-G
F-AA
S
(NS)
NAA
Rep
eata
bilit
y & m
er
y repo
rted;
Interference by o
ther
met
als assessed
Rep
eata
bilit
y&rc
cova
yrep
ortc
d
Stan
dard
additions; m
atrix
mat
chin
g unsuccessful. used s
erum
-bas
ed st
anda
rds.
Prec
ision
. recovery &
long
term
repeatability reported.
Sero
nom
, Lan
onor
m - i
n ag
reem
ent; Seronorm ur
ine -
low
Sero
norm
- in
agre
emen
t; NEl
S bo
vine
seru
m - d
eter
min
ario
n too
low
Com
paris
on w
ith W
AC
refe
renc
e method with
AD
PC ex
tract
ion.
Sero
nonn
- in
agre
emen
t
Doc
umen
t full m
etho
ds de
velo
pmen
t.
Mdt
iele
men
t ana
lyse
s from 3
labs u
sing
Fla
me-
, GF-
. and
Z-GF-AAS.
IB-A
ES and
NA
A.
Whi
ch m
etho
ds us
ed fo
r Ni are n
ot discussed.
I+
0
P
*Val
ues an
my
re
pone
d by
auth
ors i
n m
ass c
on
me
on
. T
best
hav
e been c
onve
ned to
subs
tanc
e cm
cuud
on based
on an
atom
ic m
ass o
f Ni
of 5
8.69
. t S - b
lood
seru
m. P - b
lood
pla
sma,
B - w
hole
blo
od. U - u
rine
* Hx - m
edica
l history. Rx -
med
icat
ions
. Tob
. - tob
acco
use, O
ccup
. - oc
cupa
tion
1 (+) a
pare
ntly
adeq
uate
, (-)
poss
ibly
inad
equa
te. N
S - n
ot sp
ecifi
ed. SS
- stai
nles
s ste
el
W 9
362 COMMISSION ON TOXICOLOGY
3. PREANALYTIC SOURCES OF VARIATION
I t i s not y e t known whether N i i s e s s e n t i a l f o r human h e a l t h , o r t h e l i m i t s w i th in which i t s concen t r a t ion i n blood plasma may be r e g u l a t e d i n c h r o n i c exposure. Th i s l e a v e s open t h e q u e s t i o n of whether there i s an expected c o n c e n t r a t i o n t h a t i s h o m e o s t a t i c a l l y r egu la t ed , and t h e e x t e n t t o which concen t r a t ion of N i i s determined by i n d i v i d u a l c i r cums tances . However, ample evidence i s p r e s e n t e d below t o demonstrate t h a t i nc reased N i i n t a k e occur s f o r a v a r i e t y of r easons , and can i n c r e a s e c o n c e n t r a t i o n s i n b lood and u r i n e . Thus a l l f a c t o r s p o t e n t i a l l y a f f e c t i n g N i e x p o s u r e s h o u l d i n p r i n c i p l e be documented f o r t h e r e f e r e n c e popu la t ion . Furthermore, f a c t o r s such as age , s e x and g e n e r a l h e a l t h may have p o o r l y unde r s tood e f f e c t s on N i c o n c e n t r a t i o n s , and m u s t be c a r e f u l l y r epor t ed .
Age and sex A p a r t f rom p o s s i b l e d i f f e r e n c e s due t o p r e g n a n c y and
p a r t u r i t i o n (see below) , sex d i f f e r e n c e s i n serum N i c o n c e n t r a t i o n have no t been demonstrated. Hohnadel e t a l . [211 r e p o r t e d h i g h e r c o n c e n t r a t i o n s of N i i n sweat from females [131 f 65 pg/L (2.23 f 1.11 pmol/L); n=15] than from males [52 f 36 ( 0 . 8 9 f 0 . 6 1 ) ; n=331 as measured by flame atomic a b s o r p t i o n spec t romet ry . However, t h e s e va lues are c l ea r ly t o o high, and were compromised by t h e t echn iques a v a i l a b l e a t t h e t i m e ; N i c o n c e n t r a t i o n s i n sweat o f 1 0 h e a l t h y people ( sex not s p e c i f i e d ) has subsequent ly been r e p o r t e d t o be 4 . 4 f 2.5 pg/L (75 f 37 nmol/L) [ 1 2 ] . Furthermore, t h e r e i s no r e l i ab le in fo rma t ion on age e f f e c t s , p a r t i c u l a r l y i n t h e extreme p a e d i a t r i c and g e r i a t r i c p o p u l a t i o n s . No s i g n i f i c a n t d i f f e r e n c e s i n N i i n serum o r blood of h e a l t h y a d u l t s were found between groups over and under 40 y e a r s of age [ 9 1 . U n t i l t h e s e f a c t o r s are i n v e s t i g a t e d , r e f e r e n c e p o p u l a t i o n s should be well c h a r a c t e r i z e d wi th r e s p e c t t o t h e i r age and sex d i s t r i b u t i o n .
Geographic location A number of f a c t o r s i n the human environment determine exposure
t o N i , and t h e s e must be r e p o r t e d i n any pu rpor t ed r e f e r e n c e s t u d y . N i i n s o i l v a r i e s between about 0 . 0 9 and 9 pmol/g depending on g e o l o g i c a l f a c t o r s , w h i l e v a r i o u s u n p o l l u t e d water s u p p l i e s g e n e r a l l y have N i c o n c e n t r a t i o n s i n t h e range 0.02 t o 0 .85 pmol/L [ 2 2 ] . Water s u p p l i e s i n i n d u s t r i a l l y p o l l u t e d areas can have more t h a n 20 pmol/L. The l a t t e r can i n c r e a s e t o t h e mg/L r ange i n waters. I n t h e U.S.A., r u r a l a i r t y p i c a l l y c o n t a i n s N i a t a c o n c e n t r a t i o n of 0 . 1 0 nmol/m3, wh i l e i n urban a i r t h e c o n c e n t r a t i o n i s about 0 .43 nmol/m3 [23 , 2 4 1 . V a l u e s u p t o 2 . 9 nmol/m3 occur i n some i n d u s t r i a l c e n t e r s [ 2 4 ] . Hopfer e t a l . [17] have compared serum N i c o n c e n t r a t i o n s i n h e a l t h y h o s p i t a l workers i n H a r t f o r d , CT, U.S.A. (n=43) and Sudbury, Ont. , Canada (n=22) . T h e Sudbury cohort l i v e s i n a community of n i c k e l m i n e s and smelters, and i s exposed t o h ighe r concen t r a t ions of N i i n t a p water and s o i l . These people had h ighe r c o n c e n t r a t i o n s of N i i n t h e i r se rum [ 0 . 6 f 0.3 pg/L ( 1 0 f 5 nmol/L) i n Sudbury versus 0 . 2 f 0 . 2 pg/L ( 3 . 4 f 3.4 nmol/L) i n Hartford, p C 0.051.
Occupation Because of t h e h i g h worldwide p r o d u c t i o n and u s e of N i
compounds, occupa t iona l exposure i s widespread. Highest exposures occur i n N i r e f i n i n g and e l e c t r o p l a t i n g , arc welding and Ni-Cd s t o r a g e b a t t e r y p roduc t ion . I n d u s t r i a l exposure a l s o o c c u r s from t h e product ion of N i a l l o y s , i t s use as a c a t a l y s t f o r hydrogenation of o rgan ic compounds, as a f l u i d c a t a l y s t i n t h e petroleum i n d u s t r y , as a pigment f o r ceramics and g l a s s e s , as a mordant f o r dyeing c l o t h , and i n e l e c t r o n i c components and magnetic t a p e s [23, 2 4 1 . A s tudy of 8 workers exposed t o N i compounds i n t h e workplace r evea led a N i c o n c e n t r a t i o n o f 1 1 . 5 pmol/m3 i n a i r , whereas 103 we lde r s b r e a t h e d a i r wi th a mean N i con ten t of 1 . 6 pmol/m3 [ 2 5 1 . I n t h e
Measurement of total nickel in body fluids 363
f i rs t group, high concen t r a t ions of N i i n u r i n e c o r r e l a t e d w i t h high c o n c e n t r a t i o n s i n blood; no such c o r r e l a t i o n was found i n t h e w e l d e r s . Occupa t iona l exposure can t h e r e f o r e have s i g n i f i c a n t though u n p r e d i c t a b l e e f fec ts on N i c o n c e n t r a t i o n s i n b i o l o g i c a l f l u i d s , and must be w e l l documented.
Diet A major source of N i exposure i s d i e t a r y , account ing t y p i c a l l y
f o r a t least 1.7 p o l / d a y of i nges t ed N i [ 2 ] . However, c e r t a i n d i e t s can r e s u l t i n c o n s i d e r a b l y h i g h e r i n t a k e s . While meats have N i con ten t s i n t h e range 1 . 0 t o 7.2 nmol/g, t h e corresponding range i n f r u i t s and v e g e t a b l e s i s 0 . 9 t o 150 nmol/g [ 2 2 1 . T h e r e f o r e , v e g e t a r i a n diets w i l l lead t o h ighe r N i i n t a k e . Nuts ( e . g . cashews 85 nmol/g), sp inach (150 nmol/g d ry we igh t ) , t ea l e a v e s (77 nmol/g f r e s h weight) [ 2 2 ] , cocoa (167 nmol/g) , soy p roduc t s and oatmeal [261 a r e e s p e c i a l l y high i n N i . Diets r i ch i n these foods can cause high r a t e s of u r i n a r y e x c r e t i o n [261 .
Tobacco and alcohol C i g a r e t t e s each con ta in 34 t o 105 nmol of N i l and 10 t o 20 % i s
r e l e a s e d i n i n h a l e d smoke [ 2 3 ] . The d a i l y i n h a l e d dose of N i has been e s t i m a t e d t o be 68 nmol f o r a smoker of 20 c i g a r e t t e s p e r day [ 2 ] . T h i s i s probably i n s i g n i f i c a n t i n r e l a t i o n t o i n t a k e from food, d r i n k i n g water and o t h e r s o u r c e s . However, smoking may a l s o i n c r e a s e t h e uptake of N i and C r from o t h e r s o u r c e s i n exposed workers, g i v i n g r i se t o e l e v a t e d u r i n a r y l e v e l s f a r i n e x c e s s of t h o s e a c c o u n t a b l e f o r by metals i n h a l e d from t h e c i g a r e t t e s themselves [ 2 7 1 .
Because of t h e f e w r e l i a b l e measurements of c o n c e n t r a t i o n i n serum t o d a t e , there i s i n s u f f i c i e n t information t o a l low comment on t h e e f f e c t s of a l c o h o l consumption on N i s t a t u s . The numerous e f f e c t s of c h r o n i c a l c o h o l consumption on g e n e r a l p h y s i o l o g y , h e a l t h , n u t r i t i o n and body composition d i c t a t e c a r e f u l documentation of p a t t e r n s of a l c o h o l u se of any r e f e r e n c e p o p u l a t i o n . The N i c o n t e n t of b e e r and wine i s g e n e r a l l y i n t h e range 0.17 t o 3.4 pmol/L [ 2 2 ] , a l though t h e d i u r e t i c e f fec t of i n t a k e of l a r g e volumes of these beverages on N i e x c r e t i o n i s not known, and t he i r impact on N i concen t r a t ions i s t h e r e f o r e hard t o p r e d i c t . Health status
A number of sys t emic d i s e a s e s may a l t e r N i c o n c e n t r a t i o n s i n body f l u i d s . P a t i e n t s i n r e n a l f a i l u r e have impaired e x c r e t i o n of N i . C h r o n i c h a e m o d i a l y s i s compounds t h e p r o b l e m , a n d hypernickelaemia i n s u c h p a t i e n t s i s w e l l documented. S e v e r a l of t h e more r e l i ab le r e p o r t s of N i c o n c e n t r a t i o n s i n serum of h e a l t h y a d u l t s have included comparative d a t a on haemodialysis p a t i e n t s [11, 1 7 , 1 8 , 2 8 1 . V a l u e s i n t h e r e n a l p a t i e n t s were 3.71 f 1.54 (63.2 f 26.2) versus 0.44 f 0.18 (7.5 f 3.1) pg/L (nmol/L) [ill, 5.4 f 2.1 (92 f 36) versus 0.3 f 0.2 (5.1 f 3.4) pg/L (nmol/L) [281 , 7.2 f 2.2 (123 f 37) versus 0.2 It 0.2 (3.4 f 3.4) pg/L (nmol/L) [171 , and 6.38
& 3.36 (108 f 57.1) versus 0.14 f 0.09 (2.38 f 1.53) pg/L (nmol/L) [ 1 8 ] . Thus i n c r e a s e s of n e a r l y 50-fold may occur i n a p o p u l a t i o n undergoing d i a l y s i s . A c u t e myocardial i n f a r c t i o n i s a s s o c i a t e d w i t h an i n c r e a s e i n serum N i i n t h e fol lowing 72 h [peak, 3.0 f 3.4 (51 f 58) pg/L (nmol/L) versus 0.28 f 0.24 (4.76 f 4.08) pg/L (nmol/L) i n h e a l t h y a d u l t s ] [ 9 ] . The same s t u d y a l s o no ted h i g h e r v a l u e s i n u n s t a b l e angina p e c t o r i s [peak concen t r a t ion 1.4 f 0 . 9 pg/L (24 f 15 nmol/L) J , Ch r i s t ensen & Pedersen [ 1 2 ] found h i g h e r v a l u e s i n serum N i i n 10 rheumatoid a r t h r i t i s p a t i e n t s [0.64 f 0.3 pg/L (11 f 5.1 nmol/L)] compared t o 10 h e a l t h y c o n t r o l s [0.25 f 0.1 pg/L (4.25 f 1.70 nmol /L) ;p< 0.0251. The same s tudy a l s o r epor t ed inc reased N i i n u r i n e [2.26 f 1.8 pg/L (38.4 f 30.6 nmol/L) versus 0.82 f 0.5 pg/L (13.9 f 8.5 nmol/L)] and sweat [10.6 f 6.7 pg/L (181 f 114 nmol/L) versus 4.4 f 2.5 pg/L (75 f 43 nmol/L)] , b o t h s i g n i f i c a n t a t t h e l e v e l of p < 0.025.
364 COMMISSION ON TOXICOLOGY
Other p u t a t i v e medical causes of hypernickelemia a r e poorly documented and a r e found i n publ ica t ions repor t ing highly suspect 'normal' values; f o r a c r i t i c a l discussion see Versieck & Cornel is [ 2 9 ] . P a r t u r i t i o n may lead t o modest increases i n N i i n maternal serum, perhaps due t o r e l ease of N i from t h e p l acen ta . E a r l i e r su rp r i s ing r epor t s of dramatic increases [ 3 0 ] , however, have been re fu ted by the more r e l i a b l e data of Nomoto e t a l . [311 , who report concentrations of 2 . 1 f 0 . 9 0 pg/L (36 f 15 nmol/L) i n heal thy adul t women versus 2.85 f 0 . 6 7 pg/L ( 4 9 f 11 nmol/L) 60 min post partum. There i s l i t t l e r e l i a b l e da ta t o support proposed changes i n N i s t a t u s i n d i abe te s , hepa tob i l i a ry d i sease , cancer o r i n f e c t i o u s processes . Urinary t r a c t i n f e c t i o n s , o f t en overlooked, should always be diagnosed a t t h e time of co l l ec t ion and, i f present t he sample should be discarded.
Medication and iatrogenic exposure Cer ta in medicat ions can a f f e c t N i concen t r a t ions i n body
f l u i d s . Disulfiram increases N i uptake by che la t ing it i n t he gut [ 2 4 ] . Oral cont racept ives were considered a p o t e n t i a l source of d i f fe rences i n serum N i concentrat ions among women [ 2 1 1 . Although the s tudy repor ted no s i g n i f i c a n t d i f f e rences , it was conducted without t he benef i t of present technology, and concentrat ions above 35 nmol/L were found i n both groups. Contamination of intravenous f l u i d s p re sen t s a r i s k of t r i g g e r i n g a l l e r g i c r e a c t i o n s t o N i ; concentrat ions of up t o 1 . 7 pmol/L N i have been found i n var ious l o t s of commercial human serum albumin [ 3 2 ] . Such i a t r o g e n i c exposure might account f o r t he high concentrat ions i n hosp i t a l i zed p a t i e n t s undergoing invasive t reatment , f o r example t h e increases t h a t were noted i n c a r d i a c p a t i e n t s . Leaching of N i from orthopaedic prostheses i s a po ten t i a l ly ins id ious route of exposure [l, 33, 3 4 1 . Dental prostheses m u s t a l s o be considered. A 30-fold r i s e i n concentration of N i i n s a l iva , a t t r i b u t a b l e t o leaching from denta l pos ts of white gold, was found i n a p a t i e n t with extensive o ra l surgery [351. Corrosion of pacemaker wires i s another po ten t i a l source of N i [ 2 4 1 .
Seasonal and diurnal variation Variat ion i n exposure may be seasonal . In one repor t , t he N i
content of urban a i r rose from 0 . 2 9 nmol/m3 i n summer t o 0.43 nmol/m3 i n $ t he winter [ 2 4 1 , probably through an inc rease i n t h e burning of f o s s i l fue l s i n t he winter months. Leach e t a l . [ 9 1 found no d iu rna l v a r i a t i o n i n serum N i i n two vo lun tee r s . Although concentrat ions i n u r ine f luc tua ted , they d id not appear t o do s o s y s t e m a t i c a l l y . I n t h e absence of f u r t h e r d a t a on c i r c a d i a n v a r i a t i o n , s tandard ized time of c o l l e c t i o n ( e . g . e a r l y morning, f a s t ing ) i s recommended.
4. SAMPLE COLLECTION FOR Ni MEASUREMENT
Preparation of skin Carefu l washing of t h e sk in t o remove a i r -borne dus t i s
mandatory, i n o rder t o avoid contamination. A f i n a l wash with e thanol and drying by evaporation has been recommended [ E l . The washing procedure a l s o serves t o remove sweat from t h e sk in . The N i concentration i n sweat i s about 5 times [ I21 o r even 1 0 t o 20 times t h a t of ur ine [ 2 4 ] . Contaminant N i , and a l s o Cu, Zn and Pb, were found i n sweat co l lec ted i n a sauna [ 2 1 ] . Evaporation increases the concentration of N i on the skin surface [361. Similar ly , t he person taking t h e sample should wear p l a s t i c gloves t o avoid contamination from sweat on the hands, and the gloves should not be of t he t a l c - powdered va r i e ty .
Measurement of total nickel in body fluids 365
Selection of needle Recognizing t h e po ten t i a l f o r leaching N i from s t a i n l e s s s t e e l ,
t he IUPAC reference method f o r N i i n serum recommends t h e use of Teflon o r polyethylene cannulae f o r blood c o l l e c t i o n 1 3 7 1 . This p rac t i ce seems w e l l founded, and most s tud ie s i n recent years have adopted t h i s prac t ice (see Table 1 ) . Versieck e t a l . 1381 drew 20 mL por t ions of blood a t 37 O C through a steel needle (19G-11/2) a f t e r i r r a d i a t i o n . Neutron a c t i v a t i o n a n a l y s i s revea led s u b s t a n t i a l leaching of N i i n t o the sample. An addi t ion of 1.28 p o l / L t o the f i r s t por t ion decreased i n subsequent port ions, but was s t i l l 0 . 2 2 p o l / L by t h e fou r th . Clear ly these values suggest t h e need f o r extreme caution, although leaching f a c i l i t a t e d by i r r a d i a t i o n damage t o the needle cannot be ruled out i n t h i s s t u d y . Sunderman J r . e t a l . [El col lec ted blood from one arm of 2 1 people through a s t a i n l e s s s t e e l needle (Monoject; mass f r a c t i o n of N i 0 .091) and through an intravenous polyethylene cannula i n t he con t r a l a t e ra l arm. The mean N i concentration i n serum from the s t a i n l e s s s t e e l needle was 0 . 7 4 k 0.25 pg/L ( 1 2 . 6 f 4.3 nmol/L), versus 0.37 f 0.18 pg/L (6.3 f 3 .1 nmol/L) with t h e cannula. The mean pa i red d i f f e rence was 0.38 k 0.23 pg/L ( 6 . 5 f 3.9 nmol/L). Blood from the cannula was co l lec ted i n t o ac id washed p l a s t i c syringes, whereas blood from t h e needle was co l lec ted i n t o blue-stoppered "metal-free" Vacutainer tubes (Becton- Dickinson), s o it was r e a l l y the e n t i r e co l l ec t ion system t h a t was being compared and d i f f e rence cannot be a t t r i b u t e d t o t h e needle a lone . Col lec t ion of cerebrospina l f l u i d with s t a i n l e s s s t e e l needles was shown t o r e s u l t i n excessive contamination precluding ana lys i s f o r N i [391. Platinum needles with gold-plated Luer hubs have been f ab r i ca t ed f o r N i measurement i n serum samples [ 4 0 1 , but a r e q u i t e expensive and not gene ra l ly a v a i l a b l e . S i l i c o n i z e d needles may i n some cases be s u f f i c i e n t l y hydrophobic t o prevent leaching of N i i n t o blood [29]. However, extreme caut ion m u s t be exe rc i sed i n r e l y i n g on s i l i c o n i z a t i o n . For example, Becton- Dickinson s ingle-use hypodermic needles ( e . g . No. 51751, o f t e n descr ibed by ana lys t s a s s i l i con ized , a r e a c t u a l l y s i l i con-coa ted only on the outs ide, f o r lubr ica t ion and comfort of the p a t i e n t .
Important exceptions t o the prudent u s e of p l a s t i c cannulae a re t o be found. Bro e t a l . [161 compared concentrations of N i i n blood s e r u m of 2 0 hea l thy a d u l t s a f t e r c o l l e c t i o n i n t o acid-washed polye thylene sy r inges through e i t h e r s t a i n l e s s s t e e l sy r inges (Terumo; 18 G , s i l i con ized ) or 1 7 G o r 18 G t e f l o n cannulae. The f i r s t 5 mL of blood was discarded. Confidence i n t e r v a l s of 95 % f o r d i f f e rences between t h e samples from the needle and e i t h e r cannula were ca lcu la ted , and no s ign i f i can t d i f fe rences were found. A mean N i concentration of 0 . 4 pg/L (6 .8 nmol/L) was reported [range 0 . 2 t o 0 .5 pg/L (3.4 t o 8 .5 nmol/L) 1 ; a s imi l a r value was found with an open c o l l e c t i o n system ( t e f l o n cannula t acid-washed polyethylene t u b e ) . Nixon e t a l . [ 1 8 ] employed a c l a s s 100 clean l a b and leached a l l p l a s t i c labware with n i t r i c ac id . Blood was drawn through a s t a i n l e s s s t e e l needle (Becton-Dickinson No. 5175) a f t e r discarding the f i r s t 3 mL. The N i concentrat ion i n serum obtained by these authors , 0 . 1 4 f 0 . 0 9 pg/L ( 2 . 4 f 1 . 5 nmol/L), i s among t h e lowest values ye t reported. The use of a f lushed s t a i n l e s s s t e e l needle remains t o be re-evaluated under t h e otherwise r igorous condi t ions employed by t h i s group.
Blood collection General awareness of vascular physiology i s needed t o c o l l e c t
r e l i a b l e samples. Changes i n pos ture may a f f e c t i n t r a v a s c u l a r volume. Pooling of blood i n the lower ex t remi t ies on s tanding can cause a 1 0 % inc rease i n non-d i f fus ib le components i n t h e upper ex t r emi t i e s [361, a s well a s an increase i n apparent haematocri t . Because a major f r a c t i o n of N i i s bound t o t h e macroglobulin nickeloplasmin, a s w e l l a s t o albumin and o ther p ro te ins [ 2 4 1 , an
366 COMMISSION ON TOXICOLOGY
increase i n these non-diffusible components r e s u l t s i n an apparent i n c r e a s e i n concen t r a t ion of N i . Furthermore, because t h e concentrat ion of N i i n whole blood i s severa l t i m e s t h a t i n serum [a], i n c r e a s e s i n haematoc r i t w i l l b e r e f l e c t e d i n h igh concent ra t ions i n blood. Application of a tourn ique t can force water from t h e vascular space, f u r t h e r i nc reas ing t h e apparent concentrat ion of non-diffusible components [ 3 6 1 . Blood should be co l lec ted w i t h t he donor rec l in ing and use of a tourniquet should be avoided. Use of a l a rge r gauge needle o r cannula (preferab ly 1 9 gauge o r l a rge r ) w i l l reduce the volume of the sample contact ing the surface, and w i l l minimize haemolysis from turbulent flow when blood components ( e .g . serum, red c e l l s ) a r e t o be separated. Tourniquets and rap id withdrawal of blood a l s o cont r ibu te t o haemolysis. The IUPAC Reference Method [ 3 7 ] c a l l s f o r co l l ec t ion v i a polyethylene cannula (d iscard ing a t l e a s t t h e f i r s t 2 mL) i n t o a polypropylene sy r inge and t r a n s f e r t o a po lye thylene t u b e . An appea l ing a l t e r n a t i v e , dropwise c o l l e c t i o n d i r e c t l y i n t o t h e tube under pa r t i c l e - f r ee a i r flow, may not be p r a c t i c a l w i t h any but t he most dedicated volunteers .
Blood should be allowed t o c l o t under s tandardized condi t ions ( e . g . 45 min a t 2 0 "C) and serum separated a t 900 x g f o r 15 m i n . Prolonged c l o t t i n g time, excessive cen t r i fuga l forces and delays i n s epa ra t ing serum from t h e c e l l p e l l e t w i l l a l l c o n t r i b u t e t o haemolysis. The u s e of plasma a f f o r d s immediate and g e n t l e r separa t ion , but addi t ion of ant icoagulants t o t h e c o l l e c t i o n tube probably poses insurmountable d i f f i c u l t i e s f o r a n a l y s i s a t t h e u l t r a t r a c e l e v e l . Heparin i s a po lye lec t ro ly t e t h a t av id ly binds d iva len t ca t ions , including N i [41], and i s d i f f i c u l t t o pu r i fy . Other Ca2+ scavengers ( c i t r a t e , oxalate , EDTA, f l uo r ide ) pose a high r i s k of contamination [36].
Washing of labware A l l surfaces coming i n t o contact w i t h the sample f o r u l t r a t r a c e
ana lys i s m u s t be scrupulously cleaned. This i s of course t r u e when the ana ly t e i s N i i n body f l u i d s . A p r a c t i c a l i l l u s t r a t i o n of po ten t i a l p i t f a l l s i s given by Nixon e t a l . [ l a ] . Forty-seven serum samples were co l lec ted i n acid-washed Monovette syr inges and handled throughout f o r ana lys i s by ET-AAS i n a c lean room. However, they were e i t h e r t r a n s f e r r e d i n t o non-acid-leached Sa r s t ed t tubes , o r a l iquoted w i t h non-acid-leached p i p e t t e t i p s . Against a reference concentration of N i of 0 . 1 4 f 0 . 0 9 pg/L ( 2 . 4 f 1 .5 nmol/L) f o r t he lab, f a i l u r e t o leach the tubes produced a value of 0.93 f 0 . 6 4 pg/L ( 1 6 f 11 nmol/L) (n=22), whereas unwashed p i p e t t e t i p s gave a value of 3 .3 f 4 . 0 pg/L ( 5 6 f 68 nmol/L) (n=25). Moody and Lindstrom [42] have compared the amount of N i leached from several types of p l a s t i c and Teflon, by hydrochloric and n i t r i c ac ids (Table 2 ) . Although n i t r i c ac id always leached more N i l t he r e l a t i v e e f f i c i e n c i e s of the two ac ids d i f f e r e d w i t h t h e mater ia l , and t h e au thors recommend sequent ia l use of both ac ids . They have a l s o recommended t h a t f i n a l r i n s i n g and soaking should be w i t h t h e pu res t water a v a i l a b l e , although reagent-grade ac ids a r e adequate f o r t h e leaching s t e p s . They f u r t h e r recommend s torage of vesse ls f o r severa l weeks ( u n t i l u s e ) f i l l e d with u l t rapure water, t o allow t h e cleaning process t o continue ,
Guidelines f o r cleaning labware f o r t r a c e and u l t r a t r a c e metal ana lys i s can be found i n many excel lent sources. Some a r e compiled i n Table 3 . A f e w make spec i f i c comments about N i l b u t one i s bes t advised t o follow general p r inc ip les and e r r on the s ide of caut ion. Any of t hese precaut ions can be relaxed only when t h e impact of t h e i r omission on t h e a n a l y t i c a l r e s u l t has been thoroughly evaluated.
Ta
ble
2 -
Am
ount
o
f N
i le
ac
he
d fr
om
pla
sti
cs
(n
mol
/rn2
) d
uri
ng
7
d
of
ex
po
sure
t
o
ac
id
(vo
lum
e fr
ac
tio
n
in
wat
er
0.5
) a
t ro
om
tem
per
atu
re
(80
OC
for
Te
flo
n).
Acid
T
eflo
n FEP
Po
lyet
hyle
ne
Poly
ethy
lene
Po
lyca
rbon
ate
oinear)
HC
l 14
0 14
0 50
50
340
270
85
120
Tabl
e 3 - So
me p
roto
cols
for c
leani
ng o
f sam
ple v
esse
ls fo
r ultr
atra
ce an
alys
is (A
R -
Ana
lytic
reag
ent g
rade
; DD
- dis
tille
d, d
eion
ized
).
0,
Ref
. ht
ende
d V
esse
l PI
E-w
ashi
ng
Acid washing
I)lw
tion
Rin
smg
hyins
8
sam
ple
s 3 ii' R
[42]
Aq
ueou
s Pl
astic
s,Tef
lon
ry
0-
0
l:l(
vh) H
Cl,
1:1(
v/v)
HN
@;
7d ea
ch,
ultr
apul
ewat
er,so
aL
wrt
icls
free
envi
ruun
ent
5' A
R;
RT (8
0°C
for T
eflo
n)
rinse
betw
een
as lo
ng as
poss
ible
P
K
14
31
Blo
od. U
rine
, Glass 1
0 plastics
No
n-i
~c -g
ent
6 m
ol/L
HC
l (A
R)
7 d each,
Disw
&io
nize
d water
Ahu
ninl
nn ov
en, v
orte
x with
x 3
1371
Po
lYeU
rylen
e
senrm
1 m
I/L H
Cl (DD)
rins
ebet
wee
n 6
mom
HCI
& w
ata be
fm u
sing
s
Hot
detergent, ta
p Co
nc. HCI fu
mes
. then
1 h
m fu
mes
. D
eion
ized
waE
rx~,
then
~
dr
y~
~~
mp
oly
e(h
yle
ne
w
att7
rinSe
.rhen
di
lute
x40
mm
inaf
terd
ilutio
n u
lmw
ate
rx
2
CB
Illljs
teT,
lid
ajar
&
ioni
zedw
ater
x6
Hig
h-pd
y quertz,
Dist
illed
water, 30%
l:l(v
/v) H
2SO
4:H
NO
3 B
oilx
8h
Oua
rtZD
Dw
ater
. O
ven dry, 55
°C on T
e&n
foil
Teflo
n sto
pper
s (v
/v) H
a fur 2
d.
(bot
h anrcennated,
rinse.
boi
l 2 x 8
h.
Poln
mpy
l- my
1291 -
rins
ewit
h2xq
uam
- U
ltrap
llre)
steam
cle
an 6
-8 h
dist
illed
wat
er
[441
N
atur
al Linearpolyethylene
Wat
ers
1.5 m
om H
N@
(A
R)
7dm
irdm
um
Rin
sew
ithsa
mpl
eat
leas
tx4
1181
Plastics, T
eflm
dilute H
N@
UN
ilUSe
Tr
iply
dist
illed
water
wm
clef
reea
ir
368 COMMISSION ON TOXICOLOGY
5.
When consider ing long-term s torage of a sample, t h e ana lys t must be aware of t h e r i s k of loss or add i t ion of t h e ana ly te by adsorpt ion t o , o r leaching from, the s torage container ; water l o s s from poorly sea led o r permeable containers ; and changes i n sample homogeneity, €or instance formation of a p r e c i p i t a t e i n s tored ur ine t h a t may adsorb N i . The chance of leaching imposes t h e same cons idera t ions a s i n sample co l l ec t ion , although c r i t e r i a may be even more s t r ingen t because of prolonged contact with t h e container . Adsorpt ive l o s s e s of N i t o most p l a s t i c s a r e probably not s i g n i f i c a n t . Stoeppler [221 found no adsorpt ion of r a d i o a c t i v i t y onto the walls of polyethylene b o t t l e s when 6 3 N i was added t o f resh ly voided u r ine . No adsorpt ive lo s ses were observed over 6 months. when ur ine was spiked with small amounts of N i and s to red a t 4 O C
(container not spec i f ied) [361 . The IUPAC reference method f o r N i i n u r i n e recommends s t o r a g e o f u r i n e a t -20 O C i n screw cap polypropylene tubes , a f t e r a c i d i f i c a t i o n with n i t r i c a c i d [ 3 7 1 . Stoeppler has a l s o reported adsorption of 6 3 N i t o t h e sediment t h a t forms when ur ine i s s tored . This occurred even when t h e sample was ac id i f i ed , accounting fo r about 6% of t he N i a t pH 6 and about 1% a t pH 1 [ 2 2 1 . Sunderman Jr . [451 recommends ac id i f i ca t ion by addi t ion of 1 0 mL H N 0 3 / L ur ine and s torage a t 4 O C f o r up t o a week ( -20 O C f o r longer s t o r a g e ) . Medium- t o long-term s torage of ur ine a t -20 O C
without ac id i f i ca t ion i s preferred by some authors [ 4 6 ] .
The r a t e of water l o s s from va r ious Teflon and p l a s t i c conta iners was inves t iga t ed by Moody and Lindstrom [ 4 2 1 . Annual l o s ses var ied from 0.03 t o 0 . 0 5 % from polypropylene and 0 . 0 5 % from Teflon FEP, t o 1 . 6 t o 2 . 0 % from polycarbonate . Losses from t r ansp i r a t ion versus around the closure were not d i s t inguished . I f long-term storage i s necessary, use of well-sealed polypropylene o r Teflon vesse l s should be considered, and surface a rea f o r leaching and adsorpt ion should be minimized by avoiding conta iners t h a t a r e la rger than necessary.
STORAGE OF SAMPLES FOR ANALYSIS
6. SAMPLE PREPARATION FOR ATOMIC ABSORPTION SPECTROMETRY FOR Ni IN SERUM AND URINE By f a r t h e most widespread method a v a i l a b l e t o many
i n v e s t i g a t o r s wanting t o measure N i i s e l e c t r o t h e r m a l atomic absorp t ion spectrometry (ET-AAS); c f . Table 1. Numerous o the r methods have been reviewed [ 2 2 , 4 7 , 4 8 1 . Though d e f i n i t i v e methods based on i so tope-d i lu t ion mass spectrometry w i l l undoubtedly be developed, suggested reference l eve l s w i l l continue t o be based on ET-AAS. The requirements f o r sample processing f o r t h i s technique i n some ways d i c t a t e short-term handling and pre-storage condi t ions. For example, i f u r ine i s t o be a c i d i f i e d f o r s to rage and l a t e r ana lys i s , an ac id compatible with the ana lys i s would be chosen. A s another example, i f serum i s t o be wet ashed f o r a n a l y s i s , g r a t u i t o u s t r a n s f e r t o an intermediate s torage conta iner may be undes i r ab le . Therefore , it i s appropr i a t e i n a d i scuss ion of sampling and s torage f o r N i measurement t o consider a l s o sample preparation f o r ET-AAS ,
The e a r l i e r IUPAC reference method f o r N i i n serum and ur ine [371 involved complexation of N i i n an a c i d d iges t with ammonium pyrrolidin-1-yldithioformate fol lowed by e x t r a c t i o n wi th 4 - methylpentan-2-one. Advances i n ET-AAS have rendered t h i s extensive manipulation unnecessary. Sunderman Jr . [451 has recommended protein p r e c i p i t a t i o n with n i t r i c a c i d and hea t f o r a n a l y s i s of blood, serum, cerebrospinal f l u i d and sa l iva , and a c i d i f i c a t i o n with d i l u t e n i t r i c ac id f o r ur ine . I n general , commercially ava i lab le u l t rapure n i t r i c ac id ( e . g . J . T . Baker, Merck "Suprapur") i s adequate without fu r the r pu r i f i ca t ion [ 3 7 ] , and i n general does not cont r ibu te t o the
Measurement of total nickel in body fluids 369
blank i f used spa r ing ly [ 2 2 ] . A l i s t of e a r l i e r dry-ashing procedures , and of wet-ashing i n H N 0 3 - H C 1 0 4 mixtures has been compiled by Stoeppler [ 2 2 ] . The IUPAC re ference method f o r N i required complete d iges t ion of both serum and u r ine w i t h an equal volume of H N 0 3 : H C 1 0 4 : H 2 S 0 4 i n volume r a t i o s of 0 . 6 , 0 . 2 and 0.2, res- pec t ive ly . Decomposition i s accomplished i n open b o r o s i l i c a t e tubes with stepwise increases i n temperature over 4 . 5 h, f i n i sh ing a t 300 O C with evaporation of a l l b u t H2SO4. Andersen e t a l . 1131 d i l u t e d serum w i t h 10-3 mol/L HNO3 (volume r a t i o 0 . 5 ) i n Tri ton X-100 (volume f r a c t i o n 0 . 0 0 1 ) f o r d i r e c t ana lys i s ; Drazniowsky e t a l . 1101 simply used a d i lu t ion w i t h 20 mL/L Triton X-100 (volume r a t i o 0 . 5 ) . Urine was analyzed a f t e r d i lu t ion w i t h an equal volume of 3 mol/L HNO3 i n aqueous Tri ton X-100 (volume f rac t ion 0.005) [ 4 9 1 . Nixon e t a l . a l so used a d i luen t of mol/L H N 0 3 i n aqueous Tr i ton X-100 (volume f r a c t i o n 0.001), and found no increase i n blank values when t h r e e i n j e c t i o n s of 50 FL were made i n t o t h e furnace i n s t e a d of one. Detergent serves a s a wetting agent and reduces carbon build-up i n the furnace, and i n t hese s t u d i e s T r i ton X-100 appears free of s i g n i f i c a n t con tamina t ion . Neve r the l e s s , it mus t always be considered a s an addi t iona l source of po ten t i a l contamination. The recommendations of Sunderman Jr . [ 4 5 ] seem t h e s a f e s t , al though decreased recovery because of p rec ip i t a t ion of protein-bound N i mus t be addressed. T h i s appears t o be a t h e o r e t i c a l cons idera t ion ; a c i d i f i c a t i o n should be su f f i c i en t t o d isp lace N i from binding s i t e s on known p ro te ins . Concentrated n i t r i c ac id (50 pL) i s added w i t h vortexing t o 1 mL serum and the mixture i s heated t o 70 O C f o r 5 min i n a s toppered tube . The c l e a r superna tan t i s analysed a f t e r cen t r i fuging . Urine i s d i l u t e d w i t h two p a r t s of ca . 0 . 2 % (w/v) HNO3 and centr i fuged only i f t u rb id .
7.
T h i s d i scuss ion focuses on ET-AAS a s t h e most important and genera l ly ava i l ab le approach f o r measurement of N i i n body f l u i d s . The preceding sec t ions have dea l t w i t h pre-analyt ical considerat ions appl icable t o t h i s technique. Several t echnica l improvements i n the p a s t decade have lowered d e t e c t i o n l i m i t s below t h e presumed concentration of N i i n most or a l l body compartments. These include g r e a t e r ins t rument s e n s i t i v i t y , t h e implementation of Zeeman background cor rec t ion , and furnaces t h a t provide rap id temperature r i s e during atomization [8 , 4 7 1 .
Ni MEASUREMENT BY ATOMIC ABSORPTION SPECTROMETRY
Table 4 - Temperature programs f o r N i measurement b y Zeeman- corrected ET-AAS. Data i n columns (i) t o ( i v ) a r e from refs . [8, 13, 16, 1 8 1 , r espec t ive ly .
T ( "C) Ramp ( s ) Hold ( s ) Gas
(i) (ii) (iii) (iv) (i) (ii) (iii) (iv) (i) (ii) (iii) (iv)
loo, loo, loo, - 1, 40, 10, - 1, 20, 80, - on 140, - 140, 120 60, - 60, 1 10, - 10, 10 on 190, 206, 190, 160 30, Qo, 30, 70 5, io, 5, 5 on
Chadash -, 700 -. -, 30 -, -, -, 10 on 1200, 1250, 1100, 1500 80, 30, 80, 10 50, 20, 50, 25 on4
Dry*
Atomize 2600, - 2500, - 0, -, 0, - 5, - 5, - of& -, 27b0, 2700, 2750 -, 0, 1 ,O -, 8, 3, 5 Off
Clean 2700, 2800, -, 2800 1, 1, - 9 1 3, 2, - 9 2 on
*These steps are repeated with a second sample introduction in study (lv). *In study (i), the gas flow is not turned off but is reduced from 300 mWmin to 30 mL/min for the last 5 s at 1200 OC and for the atomization step.
370 COMMISSION ON TOXICOLOGY
Tabla 5 - Performance characteristics of several protocols for analysis for Ni in blood serum by Zeeman-corrected ET-AAS.
Ref. Serum:Diluent Sample vol. Detection Limit Within-run Long term (nmol/L) imprecision imprecision
(C.V.) (C.V.) W)
[ E l 1 :0.05 50 0.85* 3.4% at 6.8 mom 8.1% at 6.0 nmoVL
El31 1 : 1 50 1.534
[161 1 :0.05 60 8% at 12 nmol/L 11% at 12 nmol/L
[lE] 1 :0.33 2 x 5 0 1 .o$ 3.2%at17nm0VL -
*95% confidence level 43 stanclad deviations above the blank
Four studies 18, 13, 16, 181 were selected from Table 1 on the basis of i) the apparent ability of the authors to approach the putative target value, ii) the reporting of analytical performance and experimental detail, and iii) geographic diversity. These four studies from Europe [13, 161 and the U.S. [ 8 , 181 all used Zeeman- corrected ET-AAS and Perkin-Elmer instruments (either the PE 5000 or PE 3030). Similar temperature programmes were used (Table 4); there is no reason to suggest that the slight differences were of analytical significance.
The various methods of sample preparation result in different dilutions of serum, with a consequent need to introduce different volumes into the graphite furnace. Table 5 shows that when serum was deproteinized by the addition of small volumes of concentrated acid, analysis of 50 FL was feasible; with dilution, multiple injections may become necessary (cf. [18]) . Within-run and long-term imprecision with coefficient of variation in the range 5% and lo%, respectively, was generally attainable, with detection limits lower than 1.7 nmol/L.
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