Redistribution of labile plasma zinc during mild surgicalstress in the rat

EDWARD KELLY, JEFFREYMATHEW, JONATHAN E. KOHLER, AMY L. BLASS, and DAVID I. SOYBEL

BOSTON, MASS

From the Division of General

Department of Surgery, Brigham

Harvard Medical School, Boston, M

Supported by American College of

lowship (to J.E.K.), T32 DK00775

support from the Department of Sur

pital (to J.E.K.), and RO1 DK0699

Submitted for publication Septem

December 5, 2010; accepted for pu

Zinc is an essential trace element and cofactor for many cellular processes. Uptakeof ionized divalent zinc (Zn21) in peripheral tissues depends on its total content in thecirculation and onmechanisms facilitating delivery to tissues in its labile form. Under-standing mechanisms of Zn21 delivery has been hindered by the absence of tech-niques to detect labile Zn21 in the circulation. In this study, we report the use of thefluorescent zinc-binding dye (ZnAF-2) to detect changes in labile Zn21 in the circulat-ing plasma of the rat under standardized conditions, including exogenous infusionsto increase plasma Zn21 and an infusion of the chelator, citrate, to decrease labileZn21 in the plasma without altering total Zn21 content. In a model of mild surgicalstress (unilateral femoral arterial ligation), plasma levels of total and labile Zn21

decreased significantly 24 h after the operation. Ultrafiltration of plasma into high-and low-molecular weight macromolecule fractionations indicated that bindingcapacity of zinc in the high-molecular weight fraction is impaired for the entire24-h interval after induction of mild surgical stress. Affinity of the filtrate fraction wasrapidly and reversibly responsive to anesthesia alone, decreasing significantly at4 h and recovering at 24 h; in animals subjected to moderate surgical stress, this re-sponsiveness was lost. These findings are the first reported measurements of labileZn21 in the circulation in any form of mild systemic stress. Zinc undergoes substantialredistribution in the plasma as a response to surgical stress, leading to increasedavailability in lower molecular weight fractions and in its labile form. (TranslationalResearch 2011;157:139–149)

Abbreviations: C ¼ concentrate; EGTA ¼ ethylene glycine tetra acetate; F ¼ filtrate; HEPES ¼4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; Kd ¼ dissociation constant; Zn21 ¼ ionizeddivalent zinc

Z inc is an essential trace element for all living cellsand is a cofactor for many cellular processes in-cluding protein synthesis, energy metabolism,

nucleic acid synthesis, gene transcription, and pro-grammed cell death.1,2 A wealth of experimental3-6

and clinical7-9 data indicate that total levels of ionizeddivalent zinc (Zn21) in the circulation are decreased in

and Gastrointestinal Surgery,

and Women’s Hospital and

ass.

Surgeons Resident Research Fel-

4 (to J.E.K. and J.M.), intramural

gery, Brigham and Women’s Hos-

29 (to D.I.S.).

ber 30, 2010; revision submitted

blication December 13, 2010.

a variety of chronic and acute conditions associatedwith impaired immune response. The groundbreakingwork of Prasad, beginning in the 1960s, demonstratedthe biological syndrome of chronic zinc deficiency in hu-mans10-12 and the clinical response to zinc supplementa-tion.13,14 Subsequent research also has demonstrated thevalue of zinc supplementation in other zinc-avid

Reprint requests: David I. Soybel, MD, Division of General and Gas-

trointestinal Surgery, Department of Surgery, Brigham and Women’s

Hospital, 75 Francis St., Boston, MA 02118; e-mail: dsoybel@

partners.org.

1931-5244/$ - see front matter

� 2011 Mosby, Inc. All rights reserved.

doi:10.1016/j.trsl.2010.12.004

139

AT A GLANCE COMMENTARY

Kelly E, et al.

Background

Zinc, an essential trace element for living cells, is

a cofactor for diverse cellular processes. More

than 99% of Zn21 in the plasma is bound to plasma

proteins. The component that is unbound is desig-

nated as labile zinc. Investigation of zinc metabo-

lism zinc has been impeded by the absence of

a method to detect labile zinc in the circulation.

Translational Significance

This report summarizes our experiments using

ZnAF-2, a fluorescent dye that binds labile zinc.

This technique reveals new insight into Zn metab-

olism that will facilitate new treatments for zinc-

avid clinical conditions such asmalnutrition, sepsis

and shock.

Translational Research140 Kelly et al March 2011

conditions such as bone marrow transplantation,15 ure-mia,16 and neurodegenerative disease.17 To date,most zinc assays used in biological research have re-ported the total zinc content of tissue or fluids. The totalcontent of Zn21 in the plasma reflects components thatare bound to plasma proteins such as albumin and alpha-2 macroglobulin,18 constituting more than 99% of thetotal content in the plasma. The component that isloosely bound or free is designated as ‘‘labile’’ Zn21.It is this component that is accessible to meet the re-quirements for key signaling and phagocytic func-tions19-21 of cells in the circulation,22,23 includingleukocytes24 and endothelium.2 These considerationsindicate that ongoing availability of labile Zn21 in thecirculation and its delivery to peripheral tissues is a vitalfactor in the cellular response to shock, injury, andinfection. As with calcium, a divalent cation withparallel activities in biology, the availability of freezinc may not correlate directly with the total circulatingconcentration.The investigation of the mechanisms of delivery has

been impeded by the absence of a method to detectfree Zn21 in the circulation. This report summarizesour experiments using ZnAF-2, a fluorescent dye thatbinds zinc selectively, as a reporter for nanomolar con-centrations of free zinc concentration in the plasma.The present study was performed to investigate the con-centration of circulating total and labile Zn21 in a ratmodel without prior zinc deprivation under conditionsof moderate surgical stress. Elimination of malnutritionand zinc deficiency from the model enables the investi-

gation of labile zinc delivery to tissues in response tostress alone to study the alteration of zinc availabilitythat may be physiologically adaptive, as opposed tothe effects of zinc depletion. To our knowledge, theseare the first reported efforts to obtain measurements ofchanges in plasma labile Zn21 in such a clinical model.

MATERIALS AND METHODS

Reagents and solutions. Unless otherwise noted, allreagents were from Sigma-Aldrich (St. Louis, Mo).ZnAF-2 was purchased from Axxora (San Diego,Calif). Fluozin-3 was purchased from Invitrogen/Molecular Probes (Carlsbad, Calif). Ringer’s solutionsused for preliminary screening studies contained NaCl145 mmol/L, KH2PO4 2.5 mmol/L, MgSO4 1 mmol/L,4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid(HEPES)10 mmol/L, ethylene glycine tetra acetate(EGTA) 0.3 mmol/L, pH 7.4, with Ca21 and Zn21

added to maintain Ca21 at 1 mmol/L and Zn21 atdesired concentrations. Free concentrations for Ca21

and Zn21 in Ringer’s solutions containing chelatorssuch as EGTA or citrate were calculated based on theinternet-accessibleWEBMAXCHELATOR (http://www.stanford.edu/�cpatton/webmaxc/webmaxcS.htm).

Experimental animals and surgical procedures. MaleSprague Dawley rats weighing 300 g to 350 g wereused for all experiments (Charles River Labs, Waltham,Mass). For some studies, animals were purchased withcannulae (micro-renethane MRE-040, externaldiameter 0.04 inches, internal diameter 0.025 inches)preplaced in the femoral artery or vein several daysprior to blood draw to minimize acute stress. Rats werehoused using standard animal care procedures(12:12-hour light–dark cycle, food and water adlibitum). All animal care and experimental proceduresused were consistent with National Institutes of HealthAnimal Care and Use Guidelines and were approvedby the Institutional Animal Care and Use Committeeat Harvard Medical School. Rats were maintainedunder general anesthesia using pentobarbitalintraperitoneal injection. Warming pads were used tomaintain temperature.For vascular access, the lower abdomen and groins

were shaved and the femoral vessels were surgically ex-posed. Cannulation of artery or vein was performed with24-gauge IV catheters (Angiocath, BD Medical, Sandy,Utah) and were accompanied by distal ligation usingfine silk ligatures. The arterial catheter was used for in-vasive blood pressure measurement using a Spacelabsmultichannel monitor (Spacelabs Healthcare, Issaquah,Wash). The catheter was flushed with normal saline tomaintain patency and provide fluid boluses as well asfor the withdrawal of blood samples (1.5 mL at each

Translational ResearchVolume 157, Number 3 Kelly et al 141

time point) from the most proximal port (�10 cm fromthe catheter) to minimize dead space.

Screening of conditions and candidate reporters formeasuring free Zn21 in plasma. Preliminary studieswere performed to identify optimal conditions for assayand included exploration of the influence of simple saltsolutions (100 uM NaCl) containing different pHbuffers (10 mmol/L HEPES, glycine, N-methyl-D-glucamine, or mono-/bi-sodium phosphate) on res-ponsiveness of those reporters to Zn21. The resultingsolutions were plated in 96-well plates and fluorescencewas measured in a Synergy 2 microplate reader (BiotekInc, Winooski, Vt). Because content of labile Zn21 inthe plasma has been predicted to reside in the nanomolarrange or lower,25-27 Fluozin-3 and ZnAF-2 (50 nMeach) were selected for investigation based on reportsof their high selectivity for zinc and dissociationconstants (Kd) in the low nanomolar range in aqueoussolution.28-30 Among the buffers, HEPES was the onlyone that did not interfere with measurements ofreporter fluorescence in simple aqueous solution. Incontrast to anticoagulants that are also chelators(EDTA and citrate) of divalent cations, we observedthat heparin does not interfere with measurements inHEPES-buffered Ringer’s if it is placed in dilutions of1:50 or less (data not shown).

Standardization of conditions for plasma assay of labileZn21 and fractionation of samples. Plasma sampleswere collected and measured immediately, as prelimi-nary experiments demonstrated an increased variabilityof measurements in samples stored on ice. Blood spec-imens were collected from the femoral artery catheter in1.5-mL aliquots and centrifuged at 10,000 xg for10 min. The plasma fraction was immediately assayedfor labile zinc. Total zinc, total protein, and albuminwere assayed in batches at the end of the experiment.In a subset of experiments, a 500-mL aliquot of

plasma from each sample was filtered through a 10-kDa plasma filter (Amicon Ultra; Millipore, Billerica,Mass) at 14,000 xg for 15 min at 4�C. The filtrateswere retained and concentrates were resuspended inzinc-free 18-MU Milli-Q water. For measurements oflabile Zn21, whole or fractionated samples of plasma(50 mL/sample) were plated in duplicate or triplicateon black 96-well plates (NUNC; Thermo Scientific,Roskilde, Denmark). After an initial baseline fluores-cence reading (ex. 485, em. 528), ZnAF-2 (1.5 mL ofa 1-mM solution in dimethyl sulfide) was added toeach well for a final concentration of 30 nM. Sampleswere mixed by gentle shaking for 10 s, and fluorescencewas reread.

Total plasma zinc assay. Plasma total zinc was deter-mined using the Quantichrom Zinc Assay kit (BioAssaySystems, Hayward Calif) according to the manufac-

turer’s instructions. This zinc assay is a colorimetric as-say based on zinc binding to a chromogen that reports at425 nm. Results were read on the microplate reader.

Plasma albumin assay. Plasma albumin was assayedusing the BCPAlbumin Assay Kit (BioAssay Systems,Hayward, Calif) according to the manufacturer’s in-structions, and results were read at 610 nm on the platereader.Statistics. Data were analyzed using standard statisti-

cal software (SigmaStat v3.5; Systat Inc, Chicago, Ill).Continuous variables are expressed as mean6 standarderror of the mean (SEM). One-way analysis of variancewas used for statistical comparison of multiple groups,and student t test was used for the before-and-after com-parison within groups where appropriate.

RESULTS

Evaluation of fluorescent Zn21 reporters for plasmaassay. To assess the validity of using vital dyes to deter-mine the zinc concentration in solution, we evaluatedthe following candidate reporters based on their knownbinding affinities for zinc in the nanomolar range:Fluozin-3 and ZnAF-2 (both in their free acid forms).Both reporters excite in the green component regionof the spectrum (485 nm), with emissions measured at528 nm. When studied in simple HEPES-bufferedRinger’s solutions, good titration curves were obtainedfor Fluozin-3, confirming published reports that theKd for Zn2131,32 lies in the low nanomolar range.When evaluated in Ringer’s solutions Fig 1, ZnAF-2provided a reliable range of fluorescence responses(from Fmin to Fmax) in response to increases in [Zn21]from �0 nM (Ringer’s 1 500 mM EGTA) to 32 nM.When evaluated in plasma, the response curvemaintained its dynamic range (ie, the ratio of thefluorescence minimum when all labile Zn21 ischelated with excess EGTA compared with thefluorescence maximum in the presence of excessZn2133). In HEPES-Ringer’s and plasma, this ratio isabout 1:8. In addition, the curve shifted markedly tothe right (Fig 1, B), indicating that the dye is capableof monitoring changes in labile Zn21 despite thepresence of high-affinity binding within the plasma.These observations accord with published estimates oflabile zinc concentration in the plasma in the range of2-10 nM.Of note, excitation of plasma at 485 nm does yield

background fluorescence.We also found that backgroundfluorescence increases with Zn21 content, increasingfrom about 10% of signal under baseline conditions toabout 20% of signal when 100 mM of Zn21 is added toa plasma sample. As a result, all measurements requirecorrection for background for each experimental condi-tion, which was done in the studies that are reported

Fig 1. Titration curves for ZnAF-2 fluorescence measured with excitation 485 nm and emission at 528 nm. In

Ringer’s solutions (A), the dye is responsive to changes in free concentration of Zn21, over the range 0 nM to

32 nM, confirming the profound buffering capacity of the plasma for Zn21. In rat plasma (B), the same range re-

sponsiveness is observed in rat plasma with all Zn21chelated [POINT X] or in samples supplemented with high

concentrations of exogenous ZnCl2, at levels up to 16 mmol/L, indicating that responsiveness of the dye is pre-

served in plasma. In panel B, the vertical dashed line indicates the fluorescence of untreated plasma minus back-

ground. This value, approximately 1400 relative fluorescence units (RFU), corresponds with a concentration of

about 1 nM of labile zinc as predicted by the Grynkiewicz fluorescence equation.33 The arrow in panel A marks

the corresponding concentration, 1 nM protein-free Ringers solution. All data are summarized as mean RFU6SD,

N 5 3 for all data points.

Translational Research142 Kelly et al March 2011

subsequently. Of note, Fig 1,A shows that the backgroundfluorescence of dye in Ringers solution is about 4000units, which was constant throughout our experiments.However, in plasma (Fig 1, B), the difference betweenbackground fluorescence of dye in plasma and fluores-cence in response to added zinc is much less. Becauseof the difficulty of independently calibrating the dye ina complex fluid such as plasma under different circum-stances, it seemed most appropriate to summarize resultsby comparison with baseline measurements (normaliza-tion to a baseline level of 1.0). Extrapolations to quantita-tive measurements are provided in the discussionsubsequently.

Sequential measurements in plasma during exogenousinfusionsof Zn21andcitrate. We next determined whetherthe ZnAF-2 assay is capable of detecting controlledchanges in the labile Zn21 content of plasma. Ratsunderwent cannulation of both femoral veins and 1femoral artery and were divided into 3 groups (n 5 4each). In the first (control) group, only normal salinewas infused in each of the venous catheters (1 cc over10 min). In the second group (Zn21-infusion), a briefbolus infusion of ZnCl2 was performed through one ofthe venous cannulae (0.4 mg/kg, 1 cc over 10 min),whereas saline was administered through the othervenous cannulae (1 cc over 10 min). To demonstratedetection of transient changes in labile Zn21 inplasma, a third group of animals received an infusionof a Zn21 bolus infusion through 1 cannula whilereceiving an infusion of sodium citrate (30 mg/kg in1 cc, over 10 min) through the other. Citrate isa moderate affinity chelator for divalent cations

including Zn21 (Kd for Zn21 18 mM). Arterial bloodsamples were drawn before (time 0) and 5, 15, 30, and60 min after infusion for measurements of total andlabile Zn21 in plasma.Measurements of total Zn21 content in plasma, using

a commercial colorimetric assay, are shown in Fig 2 andthose of labile Zn21 are shown in Fig 3. Among controlanimals, neither total nor labile Zn21 levelswere changedsignificantly over baseline. Among the animals who re-ceived an infusion of Zn21 alone, the levels of totalZn21were elevated immediately after the infusion and re-mained elevated for the duration of the study period,slowly returning toward baseline from a peak at 5 min.In these animals, increases in the levels of labile Zn21

alsowere detected, peaking at 40% to 50%above baselineat 10 min to 20 min and then slowly declining thereafter.Among the animals receiving both Zn21 and the divalentcation chelator sodium citrate, each infused through sep-arate catheters, a similar increase and time course of ele-vation occurred in levels of total Zn21. In contrast toanimals undergoing an infusion of Zn21 alone, initialincreases in labile levels were observed before a sharp,transient downturn was detected when the metal and thechelator had time to intermix in the circulation.

Alterations in plasma Zn21 levels during moderatesurgical stress. To evaluate disturbances in circulatinglevels of labile Zn21, we performed studies in a ratmodel of moderate surgical stress. Rats underwent pen-tobarbital anesthesia, skin incision, tissue dissection,unilateral femoral artery ligation and cannulation, andblood sampling at time points A (time 0), B (1 h aftercannulation), and C (5 h after cannulation). The femoral

Fig 2. Total zinc measurements in whole plasma samples from rats

undergoing infusions of saline (5 mL over 20 min, symbol C), exog-

enous zinc sulfate (0.4 mg/kg in 5 mL over 20 min, symbol B), or ex-

ogenous zinc sulfate (0.4 mg/kg) and sodium citrate (30 mg/kg)

through separate infusion catheters (symbol;). Data points are shown

as mean 6 SEM. * 5 P , 0.05 versus control, N 5 4.

Fig 3. Labile Zn21 measurements in whole plasma samples, using

ZnAF2 fluorescence, during infusions of saline, zinc sulfate, or zinc

sulfate and citrate (same animal groups as in Fig 2). Rats underwent

infusions of saline (5 mL over 20 min, symbolC), exogenous zinc sul-

fate (0.4 mg/kg in 5mL over 20 min, symbol B), or exogenous zinc

sulfate (0.4 mg/kg) and sodium citrate (30 mg/kg) through separate in-

fusion catheters (symbol ;). Labile zinc increases sharply in the ear-

liest time points after infusion, buffered transiently with the concurrent

infusion of the chelator, citrate. Data points are shown as mean 6SEM. * 5 P , 0.05 versus control, N 5 4.

Translational ResearchVolume 157, Number 3 Kelly et al 143

artery then was decannulated and ligated, and rats wereallowed to awaken. Twenty-four hours after the start ofthe initial surgical procedure, rats were reanesthetized,and the femoral artery was cannulated on thecontralateral side to collect a final blood sample (timepoint D). Rats then were euthanized by pentobarbital

overdose and exsanguination. Rats that had beenprecannulated in the femoral artery served as controlsand were placed under anesthesia only using anidentical protocol. Measurements were obtained inboth groups (n 5 6 for each group).Summarized in Fig 4 are changes in levels of total

Zn21 in plasma in precannulated control rats undergo-ing anesthesia alone (Fig 4, A) and in rats acutely butmildly stressed by anesthesia and femoral artery cannu-lation (Fig 4, B). Although some variations were ob-served over the 24 h of study in the control group,a significant decline occurred in the total plasma contentof Zn21 in the stressed group at the 24-h time point.Summarized in Fig 5 are changes in labile Zn21. Incontrol animals, these changes were remarkably con-stant (Fig 5, A) over the period of study, whereas inthe stress group (Fig 5, B), they decreased significantlyin period D. In this 24-h interval after the initiation ofstudy, significant alterations in plasma albumin—a key serum binding protein for Zn21—were notobserved in control or stressed groups (Fig 6). Thus,in period D, the ratios of total (Fig 7) Zn21 to serumalbumin were markedly decreased (P , 0.05). Thesefindings suggest that the decrease in total Zn21 wasnot because of alterations in plasma concentration ofbinding proteins; rather, the decline in total Zn21 seemsto reflect unloading of Zn21 because of changes inplasma protein binding capacity.We then explored potential alterations in the distribu-

tion of Zn21 among serum protein fractions. Plasmaproteins were separated by filtration of whole plasmasamples (0.5 mL) into high (.10 kDa) and low (,10kDa) fractions. The volume of each fraction was ad-justed to 0.5 mL, using 18-MU water, then assayedfor total Zn21 content (200 mL aliquots). Shown inFig 8, A are measurements of total Zn21 in plasma frac-tions filtrate (filtrate [F], ,10 kDa) and concentrate(concentrate [C], .10 kDa) obtained from a group ofanimals (n 5 4) subjected to moderate surgical stress.Summarized in Fig 8, B are calculations of the ratio oftotal Zn21 in the F versus C fractions, which are usedas an index of redistribution between the two. The re-sults indicate that changes in total Zn21 content ofplasma (Fig 4) largely reflect those in the large molecu-lar weight fraction. In period D (24 h), it seems thata transfer of Zn21 content takes place from the high-molecular weight fraction to the low-molecular weightfraction.We then tested the hypothesis that surgical stress had

altered the affinity of the larger and smaller protein frac-tions for Zn21. Aliquots of reconstituted plasma pro-teins (50 mL) were mixed with 1.5-mL ZnAF-2, andthen fluorescence (Ex 485 nm/Em 528 nm) was mea-sured at baseline and after an addition of 3.5-mL aliquots

Fig 4. Total zinc measurements in whole plasma samples from animals precannulated to avoid acute surgical

stress that were subjected to anesthesia alone or animals subjected to anesthesia and a mild surgical stress. In con-

trol animals (A), no significant alterations took place throughout the 24-h experimental period. In stressed animals

(B), levels decreased progressively, becoming statistically different from baseline at 24 h (Time Point D). Data

points are shown as mean 6 SEM. * 5 P , 0.05 versus time point A, N 5 6.

Fig 5. Labile Zn21 in whole plasma samples from animal groups undergoing anesthesia alone or mild surgical

stress (same animal groups as in Fig 4). In control animals (A), no significant changes were observed during

the 24-h period of observation. Stressed animals (B) exhibited significant decrease; mean 40% below baseline

at 24 h (Time Point D). Data points are shown as mean 6 SEM. * 5 P , 0.05 versus time point A, N 5 6.

Translational Research144 Kelly et al March 2011

of Zn21 that increased the total Zn21 content in the sam-ple by an increment of 8 mM. Shown in Fig 9 are sum-maries of studies in controls (n 5 6) and animalssubjected to mild surgical stress (n5 6). Labile zinc sig-nal is shown both before and after the addition of ZnCl2.A first observation is that considerable affinity for

Zn21 exists in both the high-molecular weight (C) andlow-molecular weight (F) fractions. In the C fractionsfrom control animals (Fig 9, A), ZnAF-2 fluorescencewas not altered after an addition of 8-mM exogenousZn21, indicating a high capacity for binding of Zn21.

Fluorescence in the F fraction (Fig 9, B) alsowas also at-tenuated with an addition of 8 mM Zn21, a level thatwould be expected to saturate the reporter. A second ob-servation is that in periodsBandCvariability occurred inthe affinity for Zn21 in the F fraction in control animals(Fig 9, B) and was restored to baseline levels (period A)by the time measurements obtained in period D.Of principal note were the responses in samples taken

from animals subjected to stress. As shown in Fig 9, C,even mild surgical stress led to a significant impairmentof binding in the C fraction in all periods after the

Fig 6. Plasma albumin levelsmeasured in plasma samples of animals undergoing anesthesia alone (A) or mild sur-

gical stress (B); the same animal groupswere used as in Fig 4. No significant differenceswere observed over time in

either group, confirming that stress in both groups wasmild. The stable level of albumin concentration is consistent

with mild stress, without a significant acute phase response. Data points are shown as mean 6 SEM. N5 6.

Fig 7. Whole plasma total zinc:albumin ratio calculated frommeasurements reported in Figs 4 and 5. In the group

subjected to mild surgical stress (Panel B), a decrease of more than 50% occurs in the total zinc to albumin ratio at

24 h (Time Point D). The Control Group (Panel A) do not exhibit a significant change in the zinc:albumin ratio

throughout the experiment. Data points are shown as mean 6 SEM. * 5 P , 0.05 versus time point A, N 5 6.

Translational ResearchVolume 157, Number 3 Kelly et al 145

induction of stress. Conversely, stress seemed to elimi-nate the rapid responsiveness observed in control ani-mals and, if anything, to enhance Zn21 binding in theF fraction. Taken together, these observations are consis-tent with our hypothesis that even moderate surgicalstress leads to significant redistribution of Zn21 fromthe larger- to smaller-sized proteins. In addition, thesefindings suggest that concentrations of labile Zn21 inthe circulation remain remarkably constant, in part, be-cause of such redistribution and alterations in affinityof plasma proteins.

DISCUSSION

Prior reports of assays for free Zn21 in extracellularfluids have focused on use of dialysates.27,32 In an earlyreport,25 concentrations of Zn21 were determined inequine plasma using an enzymatic bioassay, whichrequired time for equilibration, larger volumes, andmanipulation of conditions to exclude interference byMg21. In more recent reports,34,35 a fluorometric assaybased on an early generation reporter for Zn21 (zinquin)was used to measure labile Zn21 in the micromolarrange in samples of plasma and other fluids, including

Fig 8. A, Total zinc content in high- and low-molecular weight fractions obtained from plasma of animals under-

going mildly surgical stress. Plasma was separated by centrifugal filtration across a 10-kDa filter into concentrate

(.10 kDa, gray bars) and filtrate (,10 kDa, black bars) fractions at each time point.B, Calculations for the ratio of

Zn21 content in the concentrate (.10 kD fraction) to that in the low-molecular weight fraction (,10 kD fraction).

The data indicate that, over the 24-h time course of the experiment, even mild surgical stress induces a shift of

Zn21 content from the high- to the low-molecular weight fraction. Data points are shown as mean 6 SEM.

* 5 P , 0.05 versus time point A. N 5 4.

Translational Research146 Kelly et al March 2011

cell-conditioned media. A key contribution of that re-port was the recognition that ‘‘lability’’ of the metal di-valent cation is defined in large part by the selectivity,affinity, and concentration of the reporter itself. Suchstudies were conducted on samples obtained from ex-perimental subjects that were not acutely stressed. Toour knowledge, measurements of labile Zn21 in plasmasamples have not been reported in patients who areacutely ill or animals that are under experimental stress.In plasma, background fluorescencewith excitation in

the green range is not ideal but is manageable for semi-quantitative measurements. Optimal conditions for as-say include immediate processing and use of minimalvolume additions (,3%). In addition, the reporter(�30 nM) should be used in final concentrations thatminimize, respectively, the dilution of sample and sig-nificant chelation by the reporter itself. In the studies re-ported here, use of ZnAF-2 was guided by its selectivityfor Zn21 and high fluorescence yield as well as the pres-ervation of its dynamic range even in a complex fluidsuch as plasma.If the reporter maintains its Kd for Zn

21 in a complexfluid such as plasma, then it is possible to use ZnAF-2fluorescence to calculate approximate concentrationsof free Zn21. Using the Kd (5 nM) obtained in Ringer’ssolutions, which accords well with published values,and the data obtained in Fig 1, B, we may infer a freeconcentration of Zn21 of 1 nM to 3 nM under baselineconditions (see arrow in Fig 1, A). These calculationsagree reasonably well with predictions and experimen-tal observations suggesting that unbound Zn21 levels inplasma are likely to be in the nanomolar range.26-28

An important caveat of such calculations is thatchanges from baseline fluorescence levels are not line-

arly proportional to the changes in concentrations oflabile Zn21. In the middle of the dynamic range of thedye (about 5 nM), they may be so but not toward themargins of the response curve. Thus, when baselineconcentrations increase or decrease by 40%, as shownin Figs 2 and 5, corresponding changes may occur inthe actual concentration by a factor of 2 or 3. Such dis-turbances may seem small because they occur in thenanomolar range. It must be remembered, however,that such changes in this range can initiate or terminatephysiologic or pathologic processes, including the initi-ation of pathways of apoptosis36,37 and the degradationof extracellular matrix.38 The small scales on whichsuch changes may occur should not obscure their poten-tial importance in regulating activities of circulatingcells and peripheral tissues. Nevertheless, the feasibilityof fully quantitative measurements of labile Zn21 in thecirculation awaits the development of dyes that do notrequire laborious corrections and normalizationsagainst background fluorescence.With this caveat, our studies with infusions of zinc

with or without the nonspecific chelator (sodium citrate)confirm responsiveness of ZnAF-2 to changes in labilezinc in samples taken from the circulation. With infu-sions at 0.4 mg/kg (0.12 mg for a 300-g rat 5 0.12mg/66 mg/mmol5 1.8 uM) and given an initial volumeof distribution limited to plasma (�0.2 L/kg or 6 mL fora 300g rat), wewould estimate an initial increase in totalplasma Zn21 content of 300 mM/L. Such expected in-creases in the initial plasma Zn21 content would be de-tected at the upper limit of the colorimetric assay fortotal Zn21, as was observed (Fig 2, A). The observed in-crease in ZnAF-2 fluorescence accords well with thatexpected from an exogenous addition of Zn21 to the

Fig 9. Measurements of buffering capacity for Zn21 within high- and low-molecular weight fractions of plasma

proteins taken from control animals and animals undergoing mild surgical stress. Buffering capacity was assessed

by mixing samples with ZnAF2 and measuring fluorescence before and after the addition of a standard amount (8

mM) of ZnCl2. Responses of higher magnitude to the addition of exogenous Zn21 indicate a lower buffering ca-

pacity. Panels A and B provide information about buffering for samples (high-molecular weight concentrates and

low-molecular weight filtrates) from control animals, whereas panels C and D provide information for samples

from animals undergoing mild surgical stress. A, in control animals subjected only to anesthesia, a high level

of binding capacity is observed within the high-molecular weight fraction, demonstrated as nearly identical levels

of labile zinc with (C) or without (B) added zinc. B, in control animals, a transient increase in binding capacity of

the lower molecular weight fraction is observed and restored at 24 h. C, after anesthesia and mild surgical stress,

significant decreases in binding capacity in the high-molecular weigh fraction are observed and are not fully re-

stored at 24 h. D, in the stressed animals, the rapid responsiveness is lost that was observed under control condi-

tions (panel B). Data points are shown as mean 6 SEM. * 5 P , 0.05 versus control, N 5 6.

Translational ResearchVolume 157, Number 3 Kelly et al 147

highly buffered plasma (Fig 1, B). Moreover, we showthat this reporting system can be used to monitorchanges in plasma Zn21 when high capacity chelators,such as citrate, are simultaneously introduced into thecirculation.Our studies also demonstrate that ZnAF-2 can be used

to interrogate the affinity of plasma, and its differentprotein fractions, for free Zn21. Using ZnAF-2, we con-firm that normal plasma has a marked binding capacityfor zinc and that the binding capacity largely—but notexclusively—reflects binding by largemolecular weightmacromolecules (Fig 9) such as albumin and alpha-2macroglobulin.18 We also provide evidence for bindingin lower molecular weight (,10 kD) fractions, whichbecomes proportionately more important during acutestress. Our studies indicate that changes in affinity canbe monitored in large and small molecular weight frac-tions in samples taken directly from the circulation.With characterization of the assay and its limitations,

we could investigate labile zinc distribution in a model

of mild surgical stress. The model itself involves dissec-tion, cannulation, and ligation of the femoral vessels inthe rat hind limb. This degree of surgical stress hasbeen characterized in previous studies as mild and isnot associated with elevation in circulating markers ofinjury.39 In our model, we show that plasma albuminand acute phase reactant is unchanged throughout the ex-periment. This finding confirms that alterations in totaland free levels of circulating Zn21 are not easily attrib-uted to changes in levels of important binding proteinsbut also confirm the mild nature of the surgical stressin this model. In this study, we find that even mild surgi-cal stress leads to decreases in circulating Zn21 overalland causes alterations in distribution of Zn21 among dif-ferent fractions of plasma proteins. The decline in totalzinc levels was not attributable to a concomitant declinein plasma albumin concentration, which did not changesignificantly 24 h after stress induction. The subsequentstudies using plasma fractions indicate that changes inzinc affinity of plasma proteins is the likely cause of

Translational Research148 Kelly et al March 2011

the overall decline in total Zn21 levels and may functionto regulate concentrations of labile Zn21 in the plasmathat are observed throughout the study. The results ofour experiments extend and clarify early clinical reportsthat zinc in the plasma is unloaded to peripheral tissuesduring some forms of acute stress.40

Our studies suggest that Zn21 content of plasma canshift between high affinity, high-capacity pools in high-molecular weight fractions to low affinity or labile poolsduring surgical stress. Much like the ionized calciumpool in acute calcium deficient conditions,41,42 this redis-tribution of Zn21 may occur so that it can be transferredmore easily tomeet demand in peripheral tissues.Our ob-servations also emphasize that anesthesia and moderatesurgical stresses can elicit subtle, acute responses in de-livery and clearance of metal ions from the circulation.Modification of plasma albumin metal ion(cobalt) bind-ing affinity has been shown to occur in response to organischemia.43,44 Our model characterizes the responses ofboth the total circulating zinc pool and the small labilefraction in acute stress; our results indicate that totalzinc and labile zinc both decrease in mild stress andthat zinc binding increases in the lowermolecular weightplasma fraction.Our results andother reports43,44 suggestthatmetal ionbinding has a dynamic role in themetabolicresponse to stress and can be studied independent of othermarkers. In addition, chronic zinc deficiency and zinc-responsive conditions such as chronic steroid use maybe studied in terms of labile zinc affinity.Acute changes in zinc availability have not been rec-

ognized until now as having clinical significance.Chronic Zn21 deficiency is well recognized as a risk fac-tor for infection and poor healing. Increasingly, however,it has become clear that the demand of peripheral tissuesand parenchymal cells for Zn21mayoccur in response toendocrine stimulation45 and normal physiologic activ-ity,46 as well as to oxidative stress47,48 and systemic sep-sis.24 Rapidly deployable assays for free Zn21 inextracellular fluids such as the one described here arecrucial for the development of studies to understandhow such an acute demand for Zn21 is satisfied and toidentify experimental conditions and clinical circum-stances in which the capacity of the circulation to deliverZn21 is insufficient to meet the demand. Clinical condi-tions previously identified as zinc deficiency may needto be redefined in the context of zinc use and distribution.

The authors gratefully acknowledge Dr Christopher J. Frederickson

and Dr Leonard L. Giblin for insightful review and commentary on

this series of experiments.

REFERENCES

1. Truong-Tran AQ, Carter J, Ruffin RE, Zalewski PD. The role of

zinc in caspase activation and apoptotic cell death. Biometals

2001;14:315–30.

2. Vallee BL, Falchuk KH. The biochemical basis of zinc physiol-

ogy. Physiol Rev 1993;73:79–118.

3. Hallquist NA, Khoo C, Cousins RJ. Lipopolysaccharide regulates

cysteine-rich intestinal protein, a zinc-finger protein, in immune

cells and plasma. J Leukoc Biol 1996;59:172–7.

4. Heinrich PC, Castell JV, Andus T. Interleukin-6 and the acute

phase response. Biochem J 1990;265:621–36.

5. Pekarek RS, Beisel WR. Effect of endotoxin on serum zinc con-

centrations in the rat. Appl Micriobiol 1969;18:482–4.

6. Giroux E, Schechter PJ, Schoun J, Sjoerdsma A. Reduced binding

of added zinc in serum of patients with decompensated hepatic

cirrhosis. Eur J Clin Invest 1977;7:71–3.

7. Gosling P, Rothe HM, Sheehan TM, Hubbard LD. Serum copper

and zinc concentrations in patients with burns in relation to burn

surface area. J Burn Care Rehabil 1995;16:481–6.

8. Fraker PJ, King LE. Reprogramming of the immune system dur-

ing zinc deficiency. Annu Rev Nutr 2004;24:277–98.

9. Golden BE, Golden MH. Effect of zinc on lean tissue synthesis

during recovery from malnutrition. Eur J Clin Nutr 1992;46:

697–706.

10. Prasad AS, Schulert AR, Miale A, Farid Z, Sanstead HH. Zinc,

iron and nitrogen content of sweat in normal and deficient sub-

jects. J Lab Clin Med 1963;62:84–8.

11. Prasad AS, Oberleas D, Halsted JA. Determination of zinc in bi-

ological fluids by atomic absorption spectrophotometry in normal

and cirrhotic subjects. J Lab Clin Med 1965;66:508–16.

12. Prasad AS. Importance of zinc in human nutrition. Am J Clin Nutr

1967;20:648–52.

13. Prasad AS, Miale A, Farid Z, Sanstead HH, Schulert AR,

Darby WJ. Biochemical studies on dwarfism, hypogonadism

and anemia. Arch Internal Med 1963;111:407–13.

14. Sanstead HH, Prasad AS, Schulert AR, et al. Human zinc defi-

ciency, endocrine manifestations and response to treatment. Am

J Clin Nutr 1967;20:422–31.

15. Papadopoulou A, Nathavitharana K, Williams MD,

Darbyshire PJ, Booth IW. Diagnosis and clinical associations of

zinc depletion following bone marrow transplantation. Arch Dis

Child 1996;74:328–31.

16. Mahajan SK, Prasad AS, Rabbani P, Briggs WA, McDonald FD.

Zinc deficiency: a reversible complication of uremia. Am J Clin

Nutr 1982;36:1177–83.

17. Squitti R, Gorgone G, Panetta V, et al. Implications of metal ex-

posure and liver function in Parkinson patients resident in the vi-

cinity of ferroalloy plants. J Neural Transm 2009;116:1281–7.

18. Foote JW, Delves HT. Albumin bound and alpha 2-macroglobulin

bound zinc concentrations in the sera of healthy adults. J Clin

Pathol 1984;37:1050–4.

19. Aird WC. The role of the endothelium in severe sepsis and multi-

ple organ dysfunction syndrome. Blood 2003;101:3765–77.

20. Kim KS, Rajagopal V, Gonsalves C, Johnson C, Kalra VK. A

novel role of hypoxia-inducible factor in cobalt chloride- and

hypoxia-mediated expression of IL-8 chemokine in human endo-

thelial cells. J Immunol 2006;177:7211–24.

21. Spittler A, Razenberger M, Kupper H, et al. Relationship between

interleukin-6 plasma concentration in patients with sepsis, mono-

cyte phenotype, monocyte phagocytic properties, and cytokine

production. Clin Infect Dis 2000;31:1338–42.

22. Driessen C, Hirv K, Rink L, Kirchner H. Induction of cytokines by

zinc ions in human peripheral blood mononuclear cells and sepa-

rated monocytes. Lymphokine Cytokine Res 1994;13:15–20.

23. Karl L, Chvapil M, Zukoski CF. Effect of zinc on the viability and

phagocytic capacity of peritoneal macrophages. Proc Soc Exp

Biol Med 1973;142:1123–7.

Translational ResearchVolume 157, Number 3 Kelly et al 149

24. Docke WD, Randow F, Syrbe U, et al. Monocyte deactivation in

septic patients: restoration by IFN-gamma treatment. Nat Med

1997;3:678–81.

25. Magneson GR, Puvathingal JM, RayWJ Jr. The concentrations of

free Mg21 and free Zn21 in equine blood plasma. J Biol Chem

1987;262:11140–8.

26. Maret W. Crosstalk of the group IIa and IIb metals calcium and

zinc in cellular signaling. Proc Natl Acad Sci U S A 2001;98:

12325–7.

27. Zhang P, Allen JC. A novel dialysis procedure measuring free

Zn21 in bovine milk and plasma. J Nutr 1995;125:1904–10.

28. Haugland RP. Molecular probes product information and cata-

logue. Eugene, OR: Molecular Probes, 2003.

29. Kawabata E, Kikuchi K, Urano Y, Kojima H, Odani A, Nagano T.

Design and synthesis of zinc selective chelators for extracellular

applications. J Am Chem Soc 2005;127:818–9.

30. Qian WJ, Peters JL, Dahlgren GM, Gee KR, Kennedy RT. Simul-

taneous monitoring of Zn21 secretion and intracellular Ca21 from

islets and islet cells by fluorescence microscopy. Biotechniques

2004;37:922–30.

31. Frederickson CJ, Giblin LJ, Krezel A, et al. Concentrations of ex-

tracellular free zinc (pZn)e in the central nervous system during

simple anesthetization, ischemia and reperfusion. Exp Neurol

2006;198:285–93.

32. Zalewski P, Truong-Tran A, Lincoln S, et al. Use of a zinc fluoro-

phore to measure labile pools of zinc in body fluids and cell-

conditioned media. BioTechniques 2006;40:509–20.

33. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca21indicators with greatly improved fluorescence properties. J Biol

Chem 1985;260:3440–50.

34. Komatsu K, Kikuchi K, Kojima H, Urano Y, Nagano T. Selective

zinc sensor molecules with various affinities for Zn21, revealing

dynamics and regional distribution of synaptically released

Zn21 in hippocampal slices. J Am Chem Soc 2005;127:

10197–204.

35. Kikuchi K, Komatsu K, Nagano T. Zinc sensing for cellular appli-

cation. Curr Opin Chem Biol 2004;8:182–91.

36. Kohler JE, Mathew J, Tai K, Blass AL, Kelly E, Soybel DI. Bac-

terially-generated oxidant monochloramine (nh2cl) impairs

caspase-3 activity by releasing intracellular zinc stores. J Surg Re-

search 2008;144:304–5.

37. Kohler JE, Dubach JM, Naik HB, Tai K, Blass AL, Soybel DI.

Monochloramine-induced toxicity and dysregulation of intracel-

lular Zn21 in parietal cells of rabbit gastric glands. Am J Physiol

Gastrointest Liver Physiol 2010;299:G170–8.

38. Isaksen B, Fagerhol M. Calprotectin inhibits matrix metallopro-

teinases by sequestration of zinc. J Clin Pathol Mol Pathol

2001;54:289–92.

39. Pelinka LE, Bahrami S, Szalay L, Umar F, Redl H. Hemorrhagic

shock induces an s 100 b increase associated with shock severity.

Shock 2003;19:422–6.

40. van Rij AM, Hall MT, Bray JT, Pories WJ. Zinc as an integral

component of the metabolic response to trauma. Surg Gynecol

Obstet 1981;153:677–82.

41. Bushinsky DA, Monk RD. Electrolyte quintet: calcium. Lancet

1998;352:306–11.

42. Zaloga GP, Chernow B. Themultifactorial basis for hypocalcemia

during sepsis. Studies of the parathyroid hormone-vitamin D axis.

Ann Intern Med 1987;107:36–41.

43. Sadler PJ, Tucker A, Viles JH. Involvement of a lysine residue in

the N-terminal Ni2 and Cu2 binding site of serum albumins: com-

parison with Co2, Cd2, Al3. Eur J Biochem 1994;220:193–200.

44. Bhagavan NV, Lai EM, Rios PA, et al. Evaluation of human serum

albumin cobalt binding assay for the assessment of myocardial is-

chemia and myocardial infarction. Clin Chem 2003;49:581–5.

45. Flynn A, Strain WH, Pories WJ, Hill OA, Fratianne RB. Rapid

serum-zinc depletion associated with corticosteroid therapy. Lan-

cet 1971;298:1169–72.

46. Naik HB, Beshire M,Walsh BM, Liu J, Soybel DI. Secretory state

regulates Zn21 transport in gastric parietal cell of the rabbit. Am J

Physiol Cell Physiol 2009;297:C979–89.

47. Cima RR, Dubach JM, Wieland AM, Walsh BM, Soybel DI. In-

tracellular Ca(21) and Zn(21) signals during monochloramine-

induced oxidative stress in isolated rat colon crypts. Am J Physiol

Gastrointest Liver Physiol 2005;290:G250–61.

48. Lichten LA, Liuzzi JP, Cousins RJ. Interleukin-1b contributes via

nitric oxide to the upregulation and functional activity of the zinc

transporter Zip14 (Slc39a14) in murine hepatocytes. Am J Physiol

Gastrointest Liver Physiol 2009;296:G860–7.