am j clin nutr 1992 friel 473 7

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O rig in a l R esea rch Com mun ica tions --m ethods

The ana ly s is o f s tab le iso topes in u rin e to de te rm ine

th e frac tio n a l ab so rp tio n o f z in c13

Jam es K F riel, V ern o n L N a a ke, Jr , Le la n d V M ille r , P a u l V F en n essey, a n d K M ich a e l H am b id g e

A BSTRA CT W e m easu red iso top ic enrichm ent in u rin e af-

te r o ra l an d in travenou s adm in istratio n ofstab le iso to pes o f z inc

to de term in e fraction al absorp tion (FA ) . 68Zn and 70Zn w ere

adm in is te red ora lly and in travenous ly to fo ur no rm al adu lts .

Subsequen tly , u rine and feca l sam ple s w ere co llec ted fo ran d

14 d , respec tiv ely , ashed , and passed th rou gh io n-exchange co l-

um n s to separa te z in c f rom other e lem en ts . S am ples w ere an-

alyzed by fast-a tom -b om bardm ent m ass spec trom etry . F rom 32

h onw ards the en richm en t o f 6 8Zn and 70Zn in urine dec lin ed

propo rtio na te ly so tha t FA cou ld be de te rm ined as fo llow s: FA

= enrichm ent (o ra l/iv ) X dose ( iv /o ra l) . FA de te rm ina tions from

ur ine and feces (cum ula tive exc retio n ) w ere, respectiv ely , fo r

sub jec t ZK 1, u rine 0 .79 ± 0 .03 and feces 0 .7 0 ± 0 .0 1 ; ZK 2 ,

0 .79 ± 0 .05 and 0 .6 9 ± 0 .0 2 ; ZK 3 , 0 .26 ±.0 1 a n d 0 .25 ± 0 .01 ;

and ZK 4, 0 .4 1 ± 0 .0 2 and 0 .37 ± 0 .02 . ZK 1 and ZK 2 rece ived

the ora l iso tope w hile fas ting w hereas ZK 3 and ZK 4 rece ived

the ora l iso tope w ith m ea ls. FA of z inc can b e d ete rm ined by

m easurem en t o f iso tope enrichm en t in u rin e. Am J C /in

Nutr l992 ;55 :473-7 .

KEY W O RD S Z in c, stab le iso topes, u rin e, frac tion alb-

so rp t ion

In troduct ion

Z inc m etabo lism h as b een stud ied in hum ans by us ing bo th

rad ioac tiv e and s tab le iso top es as trace rs . Jangho rban i an d Y oung

(1) , John son (2 ), T urn lu nd e t a l (3 ), and S erfass e t a l (4 ) deve loped

the m ethod of feca l m onito ring b y using stab le iso topes to de-

te rm ine the b ioav ailab ility o fz inc in hum an d ie ts. Z inc absorp -

tion is calcu la ted as the d iffe rence be tw een theo se a dm in is ter ed

and th e trace r exc re ted ineces. B ecause ofendogenous secre tion

of absorbed iso tope an d incom p le te ex cre tion o f unabsorbed

iso to pe , W astn ey and H enk in (5 ) fu r the r p ro posed a com part-

m en ta l m ode l tha t ref ine s ca lcu la tion s o fz inc abso rp tion by use

o f feca l m on ito rin g .Y ergey et a l (6 ) recen tly reported m easuring the frac tiona l

absorp tion ofan ora l dose ofca lcium from th e ana lys is o f u rine

fo r the excre tion of bo th o ra lly an d in travenou sly adm in iste red

s tab le iso topes . Th is p ro cedu re pro v ides an eas ier w ay to m easure

fractio na l ab so rp tion com pared w ith cu rren t techn iq ues req u ir-

ing long feca l co llec tio n period s and /o r m u ltip le ven ip unc tu res

(7 , 8). In the presen t repor t w e presen t a n ew m eth od fo r m ea-

su ring frac tion al absorp tion (FA ) of z inc by us ing the an alys is

o ftw o s tab le iso top es in urine sam ples tha t w ere co llec ted afte r

an ora l and in travenous do se of these stab le iso top es w ere ad

m in iste red to h um an sub jec ts.

Meth od s

A ll g lassware an d co lum ns w ere w ash ed in 0 .1 m ol n itric acid /

L be fo re use . D isp osab le p la stic con ta ine rs an d p ipe tte s (S a rsted t,

P rin ce ton , N J), E pp indo rf m icro tu bes 23 3-9501 (B io -R ad ,

R ichm ond , CA ), and cov ered tubes55 .5 4 2 and 65 /79 3 (Sars ted t )

w ere used w ith ou t b e ing ac id w ash ed . T h is equ ipm en t hasee n

fou nd to be cons isten tly free ofz inc by f lam e a tom ic-abso rp tion

spectro pho tom etry (A A S ) ana lysis o feach ba tch . T he AA S ana l-

yses o f to ta l z inc v alu es in a ll sam ples w ere done w ith a P erk in -

E lm er 503 a tom ic -ab so rp tion sp ec troph o tom ete r (P e rk in -E lm er,

No rwa lk , CT ) as descr ibed prev iously (8 ) . B ak er Instr-ana lyzed

a tom ic-absorp tion z in c s tan dards w ere used fo r th e AA S ana lyses

(JT B ak e r, P h illip sb u rg , N J) . D e ion ized d istilled w a te r (> 1

m u, M illipo re , E l P aso , TX ) and B aker-ana lyzed reagen t-g rade

hydroch lo ric and n itr ic ac ids w erese d th rou ghou t th e stu dy .

Z in c iso top es w ith v ariou s en richm en ts w ere ob tain ed as Z nO

from the S tab le Iso tope D iv isio n of O ak R id ge N atio na l L abo-

rato r ies (O ak R idge , TN ). T ab le 1 g iv es the na tu ral abu ndan ce

o f zinc iso topes (9 ) as w ell as the ir abun dance in the enriched

p repara tion s used in th e p resen t stud y . S to ck so lu tions w ere pre -

p ared as repo rted p rev iou sly (8 ) and w ere used to p rep are stan -

dard curv es fo r ana ly sis o f a ll sam ples.

T h is s tu dy w as approv ed by the ho sp ita l hum an e th ics com -

m ittee and , a fte r in fo rm ed consen t, iso to pes w ere adm in iste red

to fou r hea lthy adu lt hum an vo lu n teers . B ecause th is w o rk w as

deve lopm enta l in na tu re , w e adm in is te red d if fe ren t iso topes i

v ariou s am oun ts to each su b ject to ev alu ate p ossib le d ifferences

in iso tope abso rp tion . Su b jec t d esc rip tion , iso topes, rou tes o

adm in istra tio n , and dose s izes a re g iv en in T ab le 2 . Fo r su b jec ts

ZK 1 a nd ZK2 , ora l and in traveno us iso to pes w ere adm in iste red

sim ultaneous ly (a t 070 0) a fte r an ove rn igh t fa st and sub jects

F rom the D ep artm en t o f P ed ia tric s an d Pharm aco logy , U nivers ity

o fC o lo rado H ea lth S c iences C en tre , D enver.2 Supported in part by N IH N IA D DK D gran tRO l A M I 2 43 2 , N IH

gran t RR -69 from G enera l C lin ica l R esea rch C en te rs , and N IH C lin ical

Ma s s Spec trom etry G ran t R RO I 152 .

3 A ddress rep rin t reques ts to JK F rie l, D epartm en t o f B ioch em is try ,

M em oria l U nivers ity o f N ew found land S t Jo hn’s , N ew found lan d , C an-

ada , A 1B 3X 9.

R ece iv ed A pril 12 , 1991 .

A ccep ted fo r pub lica tio n Ju ly 24 , 19 91 .

47 4 FRIEL ET A L

T A B L E 1

Iso tope abundan ce o f n atu ral z in c and enriched z inc preparatio n s

Na t u r a l

Z inc iso top e abun dance*

# { 1 7 6 }Z n t

64 48 .63 0 .22 5 .83

66 27 .90 0 .24 3 .78

6 7 4 . 1 0 0 . 1 2 0 . 7 1

68 18 .75 99 .3 8 4 .6 5

70 0.62 0 .04 85.03

S V alues tak en f rom O ak R idg e N ational L abo rato ry D o cum en tation

and D eB iev re and B arnes (9 ). A v erag e atom ic w e igh t 65 .38 .

t A verage atom ic w e ig h t 67 .99 .

:1 :A v erage atom ic w eig h t 6 9 .38 .

con tinued to f ast f o r the n ex t 2 h . For su b jec ts Z K 3 and Z K 4 ,

the in trav enous iso to pe w as g iv en at70 0 and th e oral iso tope

w as g iv en in th ree equal po rtio ns, one portion at eachea l

during the day . T he oral dose w as prepared g rav im e trically and

then m easured by A A S be f ore an app rop riate am oun t w as m ea-

sured for adm in istration . T he o ral dose w as g iv en in w ater (pH

= 3 ) w ith cop ious rin s ing (4 X 10 m L ) w ith d is tilled de ion iz ed

w ater. T he in trav enous do se (1 0 m L ) w as p repared grav im e t-

rically and passed through a 0 .4 - m f ilter. T h e in trav enous do se

w as tested to en sure th at it w as py rog en f ree (L im u lus A m beocy te

Lysa te Te s t K it, W h ittak er B iop roduc ts, W alk e rsv ille , M D ). T he

in trav enous dose w as g iv en ov er a 5 -m m in te rv al th rou gh a

w in ged in f u sion se t (T erum o M ed ical, E lk to n , M D ) in se rted in

the an tecub ital v e in . A three -w ay sto pcock w asse d to w ith draw

a blo od sam ple be f ore iso to pe adm in istratio n , to in trodu ce the

iso to pe , and to f lu sh th e iso top e through the line w ith salin e (30

m L ) in an alternate sy ring e .

A ll sub jec ts w ere m ain tained on a constan t daily d ie t f or 1

w k be f ore the adm in is tration o f iso to pe and for 2 w k thereaf ter.

U nique d ie t p lan s w ere con struc ted f o r each ind iv id ual to p rov id e

a constan t daily calo ric in tak e and to m ain tain constan t w e igh t.

Iden tical m eals w ere eaten each day at the sam e tim es. T he

av erag e z inc in tak e f o r all o f th e stud y su b jec ts w as 9 ± 4

mg / d .

T w enty m illilite rs o f w hole b lood w as co llec ted b e fore the

iso to pes w ere adm in iste red (base lin e ) and ev ery m o rn ing f o r the

nex t 14 d by v en ipunc ture. B lood sam p les (15-20 m L ) w ere

tak en f rom a v e in in the an tecub ital f o ssa w ith the u se o f a

w inged in fu sion se t and dispo sab le 50 -m L p lastic sy ringes

(B ec ton D ick in son , R u therfo rd , N J). A f te r th e b lo od w as p laced

in to tw o hep arin -treated tu bes, th e p lasm a w as sep arated by

cen trif u gation at 1400 X g f o r 10 m m .

Fecal sam p le s w ere co llec ted d irec tly in to ind iv id ual, z in c -

f ree p lastic bags , w hich w ere clo sed and f roz en . A ll su b jects co l-

lec ted stoo l on ce be fo re the iso tope w as adm in iste red (base line )

and all s too ls f o r 14 d af te rw ard s.

U rine sam ples w ere co llected in 1-L ac id -w ashed bo ttle s

(N algene , R oches te r, N Y ) beg inn in g w ith the v o id v o lum e be f o re

iso to pe adm in is tration and th en po o led f o r 8 -h period s f o r the

f o llow ing 3 d . For day s 4-7 , urine w as poo led and co llec ted ov er

24 h in 3 .8 -L p las tic u rine -co llec tion bo ttle s. A pp rox im ate ly 250

m L of each co llec tion w as f roz en fo r fu ture analy sis af ter the

sam p le v o lum e had been recorded . T he tim e o f the urine sam p le

w as tak en as th e end po in t o f each co llec tion perio d .

Fecal sam p les w ere dry ed to a con stan t w e ig h t and ground

and a w eig hed portion w as p laced in tared Py rex cruc ib les and

ash ed fo r 48 h in a m uf f le f u rn ace (m ode l 1 86 , Fisher S cien tif ic ,

O ttaw a, O N ) at 425 #{ 17 6} Cs prev io u sly repo rted (8 ). A shed sam p le s

w ere tak en up in 6 m ol H C1/L and m ade up to 10 m L v o lu-

m etrically in p reparation f o r ion -ex ch an ge chrom atograp hy (10 ).

A 0. 1 m L portio n o f ashed f eces w as rem ov ed f or to tal z inc

analy s is b y A A S .

Plasm a z inc concen tration w as de term ined b y A A S bef oreash ing . Plasm a sam ples w ere then dried and w eig hed in tared

Py rex cru cib le s . T he dried p lasm a w as then treated in the sam e

w ay as f eces to prov ide a z inc ch loride so lu tion .

U rine sam p le s (1 00 m L ) w ere p laced in 600-m L beak ers and

slow ly ev aporated on a ho t p late . S m alllass beads w ere added

to each beak er to redu ce sp latte rin g and 2 m L con cen trated

n itric ac id and 1 m L H 2O 2 w ere ad ded near the end o f the ev ap -

o ration to aid in th e d igestion . D ried sam p le s w ere p laced in the

m uf f le f urnace f or 24 h at 425 #{ 176} Co e lim in ate organ ic m aterials .

A t th e end o f the d igestio n period , 0 .44 m L con cen trated n itric

acid w ere added to d is so lv e th e rem ain ing salts and the sam ple

w as brough t up to 50 m L v olum etrically w ith w ater. T w o m il-

lilite rs o f und igested urine w ere allocated f or analy sis o f to tal

z inc by A A S .

For all tissues w e chose A A S fo r to tal z in c analy s is rather than

the m eth od o f iso to pe d ilu tion becau se o f the adv an tage in cost

and our estab lished sensitiv ity and prec ision (< 1% f or g quan -

tities of z inc ) w ith th is m e thod (8). Fu rther, the use o f an ad-

d itio nal iso to pe for in v itro m easurem ents w ou ld lim it the num -

ber o f iso to pes av ailab le f or iso top e adm in istration to ou r sub-

jects.

For the p repu rif ication o f urine sam ples on ly , C he lex00

che lating re sin (B io -R ad ) w as used to separate the h igh concen -

tratio ns o f calc ium , so d ium , po tassium , and m agnes ium (m il-

ligram quan tities) f rom the trace e lem ents (m icrog ram quanti-

tie s). T h is procedure w as d ev e loped by K ingsto n e t al (1 1) f or

us e w ith seaw ater.

Che lex 100 res in , 1 00 -20 0 m esh s iz e (sod ium f o rm ) w as pre-

c lean ed by soak in g in 2 m o l HN O 3/L f or 24 h and rinsed w ith

w ater. D isp osab le Po ly -Prep chrom atog raphy co lum n s (12 m L ,

B io-R ad) w ere lo ad ed w ith 3 m L resin , w hich w as con v erted to

the N H+ f orm . E ach urine sam p le had the pH ad justed to 5 .3

± 0 .2 and w as stab iliz ed w ith 0 .07 m L 8 m o l am m on ium ace tate /

L . T h e bu f f e red sam ples w ere add ed to each co lum n w ith oc

cas ional ag itation o f th e top o f th e resin bed w ith a p lastic p ipe tte

to break up the salt lay er that o ccasionally form ed , lead ing t

reduced co lum n f low . C o lum n perfo rm ance w as no t af f ec ted b y

the larg e resin v o lum e. S od ium , calc ium , m agn esium , and po-

tassium w ere e lu ted w ith 1 m o l am m onium ace tate /L be fo re

T A B L E 2

S ub jec t and iso tope adm in istration in f o rm ation

Sub jec t , age,

and sex

iso tope

In t ravenous

iso tope

In t ravenous

Z K 1, 2 4 y, M 68Z n 4 .07 70Z n 0 .792

Z K 2 , 56 y , F 68Z n 4 .03 7 0Z n 0 .806

Z K 3, 33 y , M 70Z n 2 .98 68Z n 2 .87

Z K 4, 3 4 y , M 70Z n 2 .97 68Z n 2 .78

z 2. 4w

(_) 1 8

I - . 1 . 2z

LU 0 . 6

2 . 0 ’

1 . 6 ’

1 . 2 ’

ZN 6 8 IV

7 0 ORAL

0 4 0 8 0 1 2 0 1 6 0 2 0 0

ZINC ABSORPTION FROM URINE SAMPLES 47 5

the e lution o f trace e lements w ith 1 5 mL 2 .5 mo l HNO3/L. A ll

samples w ere taken to dryness on a ho t plate and dried overnight

in the muffle furnace to sublimate ammonium nitrate and am -

monium chlo ride (1 1 ) and w ere brought to 10 mL vo lumetrically

w ith 6 mol HC1/L.

The final step in the purification o f plasma, fecal, and urine

samples w as accomplished by ion-ex change chromatography (8 ,

10 ). B rie fly , w ashed AG-i ion-exchange res in (chloride fo rm ,

B io-Rad) w as packed in 12 -mL columns (0 .7 -mL co lumn bed

for plasma and urine , 2 .5 mL fo r feces ). A fter ac idification w ith

6 mo l HC1/L, urine , fecal and plasma samples prev iously pre-

pared in 10 mL 6 mol HC1 /L w ere applied to the co lumns .

These co lumns w ere w ashed w ith successive ly w eaker concen-

trations o fhydrochlo ric ac id and finally w ith sixo lumn volumes

o fw ater to remove the z inc . The second to fifth e luants o f plasma

and urine w ere poo led and ev aporated by us ing a ro tary evap-

o rator and brought up to 0 . 1 mL in 0 .5 mol HC1/L for analy sis

by mass spectrometry . Fo r feces the second to fifth e luants w ere

applied directly to the probe .

Zinc concentrations in all reagents and resins w ere measured

by A A S . The blank value for the to tal procedure w as de term ined

by us ing ultrapure w ater for the digestion and co lumn procedures

in equal vo lumes to that o f the urine samples . B lank v alues fo r

all de term inations w ere < 0 .5 g to tal z inc . B lank correc tio ns

were not needed fo r iso tope determ inations because calculations

show ed that alteration o f enrichment ratio s due to exogenous

addition o f zinc had < 2% affec t on final de term inatio ns o f FA .

Iso tope analysis w as performed by fas t-atom-bombardment-

induced seco ndary -ion mass spec trometry (FAB -S IMS) on a VG

70 70 E HF double -focus ing mass spec trometer (V G A naly tical,

Manchester, UK) equipped w ith an Ion Tech (London) atom

gun. The FA B sample targ e t w as made o fsilv er to e lim inate any

po tential interferences from the orig inal stainle ss s teel targ et.

Tw enty m icro liters o f sample in w ater w as placed on the targe t

and evaporated w ith a heat gun. The FAB probe was then in-

serted into the source and the sample w as analy zed. This sample

application procedure was repeated after six measurement runs,

each an average o f 100 scans,to obtain a to tal o f 10- 1 2 runs

for each sample . The mass spec trometer w as o perated in the

low -reso lution mode and ion energy se lec tion was used to dim -

mate po lyatom ic interferences (LV M iller, PV Fennessey , un-

published observ ations , 199 0 ). 70Zn-”Zn and 6SZnMZn ratio s

T IM E AFTER ADM IN ISTR AT ION O F IV ISO TO PE (HOURS)

FIG 1. U rinary enrichment w ith 68Zn (intrav enous ) and 70Zn (oral)

iso to pes for subjec t ZK3 .

L : : :RAL

0 4 0 8 0 1 2 0 1 8 0 2 0 0

T IM E AFTER ADM IN ISTRAT ION OF IV ISOTOPE (HOURS )

FIG 2. U rinary enrichment w ith 68Zn (intravenous) and 70Zn (oral)

iso to pes for subjec t ZK4 .

w ere measured to de term ine 70Zn and 68Zn enrichment. D e tails

o f the mass spec trometer operation, data acquisition, and en-

richment calculations w ere reported prev iously (8 ). Iso tope-ratio

determ inations for indiv idual samples had re lative SD sf< 5%.

D etec tion lim its fo r percent enrichment w ere 0 .04% for 70Zn

and 0 .03% for 68Zn.

FA ofthe oral dose w as calculated for plasma and urine sam-

pies according to the method of Y ergey et al (6 ) by use of the

fo llow ing formula:

FA = enrichment (oral/iv ) X dose (iv /oral)

w here iso tope enrichment w as de term ined by FA B -S IM S (8 )

N e ither the to tal sample v o lume nor the z inc concentration

required to determine FA . The determination ofFA can be done

w ith a s ing le -sample measurement because bo th iso tope enrich-

ments are analyzed s imultaneously .

An accurate determination of FA by measurement of the in-

travenously and orally adminis tered isotope in a s ing le sample

assumes that both iso topes entered the plasma at the same time .

Espec ially w ith ZK3 and ZK4 , signif icant tim e e lapsed be tw een

entrance o f intravenous and oralsotopes into the plasma. To

determine the potential inaccuracy that this time difference may

intro duce into o ur de term inations o fFA , w e fitted an ex ponential

decay func tion to each se t o f data. The resulting curve for th

oral data w as shifted by an appropriate amount of time (10 h

re lative to the iv curve and FA values w ere recalculated. This

procedure changed the FA value for ZK3 from 0 .26 to 0 .25 and

fo r ZK4 from 0 .41 to 0 .39 , a m inor change , smaller than our

measurement prec ision. For feces, FA w as determ ined by cx-

trapo lating the curve o f cumulative fecalxcre tion of iso tope

back to the y-ax is as described prev iously (12).

Results a nd d iscu ss io n

We measured abso rption of an isotope tracer g iven orally by

comparing the urinary enrichment o f this iso tope w ith that o

a second iso tope g iven intravenously . U rinary enrichment data

fo r bo th the oral and intrav enous iso topes are presented in Figure

1 for subjec t ZK3 and in Figure 2 for subjec t ZK4 . Error bars

are not included because each point on the figure is the result

o fone sample determination. In bo th figures it can be seen that

the enrichment of urine w ith the intravenous iso tope dec lines

I 2 .0C)

zLU 1 . 00

Zn 88 IV

Zn 70 ORAL

* I ± SD .

I ZK-3

A P P ROP R IA TE COLLECTION TIME. --- -.

476 FR IEL ET AL

0 40 80 120 160 200

TIME AFTER ADMIN IS TRATION O F IV IS OTOP E (HOURS )

FIG 3. Plasm a enrichm ent w ith Z n (in trav en ous) and 70Zn (ora l)

iso topes fo r sub ject ZK 4.

from its m ax im um seen in the firs t 8 -h co llec tion . T he ora l iso -

to pe reach es its h ighest en richm en t by th e 16-24 -h co llec tion

p eriod , re flec tin g the d elay due to the la te r adm in istra tion of

o ra l trace r and tim e requ ired fo r abso rp tio n . F igu re shows the

iso tope en richm en t in p lasm a fo r sub jec t ZK 4 . I t is no t su rp ris in g

th at the se d ata are sim ila r to the u rina ry en richm en t d ata becau se

the k id ney sh ou ld exc rete u rine w ith th e sam e iso top ic com -

position as the p la sm a tha t it is filte r ing (13 ) .

O n ce the slop es o f the iso to p ic d isapp ea ran ce cu rves in u rine

d ec line in a p rop ortiona l m ann er , in th is s tu dy at40 h , it is

poss ib le to calcu la te the FA (F ig 4 ). M easu rem en ts m ade by

us ing d a ta from th e firs t 36 h , b efo re the trace r fromll do ses

d isappeared a t th e sam e ra te, u nd ere stim a ted FA as w as foun d

p rev io usly fo r ca lcium (6) . The tim e at w h ich FA valu es reach

a p la teau , 40 h , is som ew ha t la ter th an w as foun d by Y ergey e t

a l (6 ) in d ete rm in in g the FA for calc ium from u rin e sam ple s fo r

an ad o le scen t (24 h) andtw o in fan ts (1 2 h). A lthou gh th is is in

par t du e to th e d elay in g iv ing th e o ral iso top e an d pe rtu rba tion

o f zin c hom eo sta sis from th e adm in istra tion of do ses, it m igh t

a lso ref lect d ifferences in age andody s ize and /o r d iffe ren ces

in th e m e tab o lism of zin c and ca lcium .

0 0 i , ,

0 40 80 120 160 200

T IME AFTER ADM IN ISTRAT ION OF IV ISOTOPE (HOURS )

FIG 4. Frac t iona l absorp t ion o f7 0Zn at each co llec tion tim e fo r sub jec ts

ZK 3 and ZK 4 .

TA BLE 3

F rac tion al absorp tion as ca lcu lated from urinary and feca l da ta fo r

ea ch sub jec t

Sub jec t U rine Feces

ZK1 0 .79 ± 0 .03 0 .68 ± 0 .02

ZK 2 0 .7 9 ± 0 .05 0 .70 ± 0 .03

ZK3 0 .26 ± 0 .01 0 .24 ± 0 .01

ZK4 0 .41 ± 0 .02 0 .37 ± 0 .02

Feca l an d urina ry m easu res o f FA are ind ep enden t m easu res

in th e sam e sub jec t du rin g the sam e tim e period . In T ab le 3

can be seen tha t FA as d ete rm ined from n ine sepa rate u rin a ry

iso to pe m easu rem en ts > 40 h are clo se to th ose m easurem en ts

based on cum ula tive feca l exc retio n o f o ra l iso to pes. P la sm a

d ata , p rov id ing an o the r m easu re o f FA , w ere av ailab le fo r sub -

jects Z K 3 (FA = 0 .3 4 ± 0 .0 2 , n = 3 ) and ZK 4 (FA = 0 .42

± 0 .07 , n = 7) an d a re reasonab ly c lose to th e ca lcu la tion s from

urine . W e atta in g ood precisio n w ith u rine becau se w e a re no t

lim ited by the am oun t th at w e ana lyze . Th is is in con tra st

p la sm a w here w e can no t tak e m ore th an a few m illiliters fo

each ana ly sis .

T he asse ssm en t o f FA by u sing en riched u rin e sam ple s isp-

p licab le ov er a w ide rang e ofsub jec t abso rp tions. S u b jec ts ZK 1

an d ZK 2 to ok the ir iso tope w h ile fa stin g and co nsequ en tly h ad

a greate r abso rp tion of the te st do se than w as seen w hen th

d ose w as con sum ed w ith a m ea l (ZK 3 and ZK 4). Th ese resu lts

fo r FA are sim ila r to tho se rep orted fo r fa sting and non fasting

sub jects(14 , 15).

Because on ly o ne v en ipun ctu re is requ ired a t th e sta rt o f the

stud y to in fuse the in traveno us iso top e , th is p roced ure is ea sie r

fo r investiga to rs an d is les s inv asive fo r the sub jec t th an is th e

collection of b loo d sam ples an d feca l sam ple s ove r an en tire

stud y p eriod . I t m ay a lso p ro ve to be m ore re liab le than to ta l

qu an tita tive feca l co llec tion s, w h ich area rd to ach iev e espec ially

in am bu la to ry stud ies and requ ire co rrec tion fo r en dog en ous

exc retio n . T he urine co llectio ns n eed n o t be com ple te and an y

sam p le tak en at the app rop ria te tim e w ill p ro v ide th e necessary

in fo rm a tion requ ired to de te rm ine FA .

These p ilo t s tud ies dem on stra te tha t the concep t o f co ncurren t

s tab le -iso tope adm in is tration p rov ides inves tig ato rs w ith a m ean s

of u sing u rin e sam p le s to m easu re the frac tion al ab so rp tion o

z inc . W e th ink th at m ore w ork is needed to im prove the ana -

lytical te chn ique , tha t is , to sh ow a sm ooth decay cu rv e a fter

iso to pe adm in istra tion (F ig s 1 -3 ). T h is va ria tion from a sm ooth

cu rve cou ld be d ue to p rob lem s durin g co llec tion , iso la tion , o

m easurem en t o f iso to p ic enrichm ent. W ork is in p rogress

e lim in ate o r reduce these sou rces o f e rro r .

In the p re sen t stud ie s w e adm in iste red tw o d ifferen t stab le

i so topes at low leve ls in sub jec ts w ho w ere fa stin g and non fastin g .

W e found consis ten t re su lts bo th w ith in the u rin e co llectio ns

and am ong feca l, p lasm a , and ur in e co llec tio ns fo r FA . T here fo re ,

w e suggest tha t u rin e en richm en t w ith stab le iso topes o f z inc

can be used to easily an d accu ra te ly m easu re the FA of an ora l

d ose o f z inc in h um an su b jects . T h is p ro cedu re p ro bab ly ha

app lica tion to th e m easu rem en ts o f o th er e lem en ts o f h um an -

nu tritio n in te res t tha t have su itab le iso top es. U

W e thank A llan Q uick and Sh erri D e lM onte fo r the ir techn ica lssis-

tance in prep ar in g the sep ara tion co lum n s.

Z IN C A B SO R PT IO N FR OM UR IN E S A M PL ES 47 7

Re fe rences

1. Jang horban i M , Y oung V R . U se o f stab le iso to pes to de term in e

b ioav ailab ility o fm inerals in hum an d ie ts u s in g the m e thod o f f ecal

m on ito ring . A m J C lin N utr 19 80 ;33 :20 2 l-30 .

2. Johnson PE . A m ass spec trom e tric m e thodf o r u se o f s tab le iso to pes

as tracers in stu d ies o f iron , z in c, and copper absorp tion in hum an

sub jec t s . J N utr 1 982 ;1 12:1414-24 .

3. T urn lu nd JR , D u rk in N , C os ta F, M argenS . S tab le iso top e stud ies

o f z in c absorp tion and re ten tio n in y oun g and e lderly m en . J N u tr

1 98 6;l 1 6:1 23 9- 47 .

4 . S e rf as s R E , T h om pson JJ, H ouk R S . I so top e ratio d ete rm ination s

by induc tiv e ly cou p led p lasm a/m ass spec trom e try f o r z in c b ioav aii-

ab ility stud ie s . A nal C h im A c t s l986 ;188 :73 -84 .

5. W astney M E , H enk in R I. C alcu lation o f z inc abso rp tion in hum ans

us in g tracers by f ecal m onito ring and a com partm en tal app roach .

J N utr l9 89 ;l 1 9:1438-43 .

6 . Y erg ey A L , V ie ra N E , C ov ell D O . R ecen t stud ie s o f h um an calc ium

m e tab o lism us ing s tab le iso tope tracers . C an J Ph y sio l Pharm aco l

1990 ;68 :973 -6 .

7. Jack son M J, Jo nes D A , E dw ards R HT , e t al. Z inc hom eos tasis in

m an: stu d ies u sing a new stab le iso top e-d ilu tion techn iq ue . B r J

N utr l9 84 ;5 1 :1 99 -20 8 .

8. Pierce PL , H am b idge K M . G oss C H , e t al. Fasttom bom bard ment

m a ss s pe ct ro m et ry f or th e de term inatio n o f z in c stab le iso top es in

bio log ical sam p les . A ria1 Chem 1987 ;59 :2034 -7 .

9 . D e B iev re P. B arnes IL . T ab le o f th e iso top ic com po sitio n o f th e

e lem en ts as d e te rm ined by m ass spec trom etry . In t J M ass S pec trom

Io n Processes 1985 ;65:2 1 1-30 .

10 . K rauss K A , M oore G E . A n ion ex change s tud ie s V . A dso rp tion o f

hy droch lo ric ac id b y a s trong base an io n ex chang er. J A m C hem

S oc 1953;75: 1 457-62 .

1 1. K ings to n H , B arn es IL , B radyJT , et al. S eparation o f e igh t tran sition

e lem en ts f rom alk ali an d alk alin e earth e lem en ts in e s tu arin e and

seawa te r w ith che lating resin and the ir de term inatio n b y g raph ite

f u rnace atom ic absorp tion sp ec trom etry . A nal C hem 1978 ;50 :2 064 -

12 . Eng l i sh JL , M ille r L V , Fenn essey PV ,Ct al. U se o f a dual iso top e

techn ique to m easure z in c absorp tion . FA S E B J 1 98 9;3 :A 1 07 9(ab str).

13 . H illm an L S , T ack E , C ov e ll DG , e t al. M easu rem en t o f true calc ium

absorp tion in p rem atu re in f an ts u s ing in trav en ous C a and o ral

“C a. Ped iatr R es l9 88 ;23 :58 9 -94 .

14 . S an dstrom B , C ederb lad A . Z inc absorp tion f rom com pos ite m eals.

II. In f lu en ce o f the m ain pro te in sou rce. A m J C lin N u tr 1980 ;3 3 :

1778 -83 .

15 . Ham bidg e K M , C asey CE , K rebs N F. Z inc . In : M ertz W , ed . T race

e lem en ts in h um an and an im al nu trition . V o l 2 . O rland o , FL : A c -

a d e mi c P r e s s , 1 98 6 : 1 - 1 37 .

am j clin nutr 1992 friel 473 7

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