MILITARY MEDICINE, 174.6:615. 2009

Noninvasive In Vivo Monitoring of Cyanide Toxicity and Treatment Using Diffuse Optical Spectroscopy in a Rabbit Model

Jangwoen Lee, PhD"; Kelly A. Keuter"t; Jae Kim, PhD"; Andrew Tran, MOt; Amit Uppal, MOt; David Mukai"t ; Sari Brenner Mahon, PhD*t ; Leopolda C. Cancio, MOt;

Andriy Batchinsky, PhDt; Bruce J. Tromberg, PhD*t; Matthew Brenner, MD"t

ABSTRACT Currently. no re liable noninvasive methods exist for monitoring the severity of in vivo cyanide (CN) to>. icity. treatment. and resulting physiological changes. We developed a broadband diffuse optical ~pectroscopy (DOS) ~yMem to measure bulk tissue absorption and scattering. DOS was used to optically monitor CN toxicity and treatment with sodtum nitnte (NaN02). To perform experiments. the DOS probe was placed on the hind leg of rabbus. A sodium CN \oluuon was mfused intravenously. DOS and concurrent physiOlogic measurements were obtained. After completiOn of CN infus1on, NaNO, was infused to induce methemoglobinemia (MetHb). During infusion of CN. blood gas mea­~urements showed an mcrease in venous partial pres\ure of oxygen (p0 ,), and following reversal. venous pO, value' decreased. DOS measurements demonstrated corresponding changes tn hemoglobin oxygenation ~tates and redox state' of cytochromc-c O'\idase (CeO) during CN mfusion and NaNO, treatment. Therefore. DOS enables detection and mom­tonng of CN tox1city and treatment with NaNO,.

INTRODUCTION Cyanide (CN)-ba~ed derivati ve~ have been used for centu­ries as poisons and chemical weapons. 1

·2 As a highly tox ic and

volatile substance, CN continues to pose a major potential chemical threat to civilians and military personnel.!·' There are numerous sources of chemical CN exposure including mili tary and industrial. More commonly, exposure to cya­nide occur-; during hou<,e fires because of the combustion of plastics such as acrylics and acrylonitriles that together with carbon monoxide, aldehydes. and soot funher worsen tissue hypoxw. resultmg 111 combustiOn-related fatalities.4 111 Studies of residential fires have found higher blood C concentrations in deceased victims than in the survivors, suggesting CN tox­icity may be a maj or component of morbidity and monality in addition to carbon monoxide poisoning, and combined expo­'>Ure appears to im;rease the toxicity of both.4 111

Currently. there 1s no rapid method to measure cyanide lev­els in the hody. Urine and whole blood analysis are the only regularly used diagnostic tests for cyanide. These methods both requi re time for collection and interpretation of results.h Accurate determination of blood CN levels are affected by both the time between exposure and specimen collection and the conditions of blood \ torage. 11 1' The cyanide mole­cule becomes unstable when exposed to temperatures higher

*I a'c:r M~~:rnbc:am ami Me!du:al Program. Becl..man La-.er lnstllule. Um­~er-11} nf Cahlilmla. 100:.! llcalth Sc•ence-. Road Ea.-.1. lr.me. CA 9:!6 12- 1475.

tD1~1'11ln ot Pulmonary ;md C'nlical Care Med1t111e. Dep;mmem ot Mcdlcme. Umvcr\IIY of Cahtnm•a lrvme. 101 The Cuy Dnve South. Bldg. 'i1. Rm. IIIJ. Orange. CA 92868.

+ln-.ti iUIC ot Surjptal Research. Brooke Army Medical Cemcr. 1400 Ra"'lcy E Chambers Avenue. f'on Sam. Houston. TX 78234

Th1s manu,cnpl wa' rccclvl!d for re' 1ew in Augu't 2008 The rcv1\Cd n~<mu":ript wa' ac~:cpted fur pubhcauon 111 February 2009.

Repnnl & Cupynghl (i) by A''oclallon of Mil nary Surgeons ot U.S .. 2009.

MILITARY MEDICINE, Vol. 174. June 2009

than 4°C: therefore. the sample~ mu o;t be handled properly and processed expeditiously to ensure the accuracy of re~ults. 1~

Thu~. the need for rapid identification of patients exp<hed to CN and the ability to cont inuously monitor response to treat­ment in a fi eld or hospital setting is vi tal.

The mechanisms of CN tox icity arc complex. 1 \.l ~> The main action of cyanide is an impairment of the abi lity of tissues to uti­lize oxygen through inhibition of cytochrome-c oxidase. which ultimately blocks adenosine triphosphate (A TP) synthes1s. 1

Cytochrome-c oxidase is the terminal oxida-.e of the mllochon­drial respiratory chain and tram.fers electrons from ferrocyto­chrome c to molecular oxygen.17 CN has a high bmdmg affinity for active 'lites on cytochrome-c oxidm.e.' '·1 ~~ When bound, the electron transpon chain is arrested w11h the near infrared (N IR) optically acti ve copper core m reduced form. preventing the donmion of an e lectron . ~ • "' Progres'>ive cytotox ic tissue hypox ia develop' quickly and immediate in tervention is nec­essary to prevent toxicity or death. 1 ~ Thu\, the need for rapid identification is compounded by the potentially large number of people with significant risks of severe injury from inten­tional or accidental mass casualty exposure events.

Until recently. CN toxici ty treatment in the United States traditionally involved the administ ration of ni trites to mduce methemoglobinemia (MetHb) followed by delivery ofthiosul­fates to promote excretionY~> ~K This is admi nistered by hold­ing gaute saturated with amyl nitri te under a patient's no'>e until intravenous acces<, is obtained. Once an 1ntravenow.. line has been established. amyl nitri te is discontmued and -.odium nitrite is administered. This oxidite<, the ferrous 10n" of hemo­globin to ferric ion. The result is methemoglobin. which '>trongly binds to cyanide a\ cyanomethemoglobin. After thi-. is completed, sodium thiosulfate is admi ni\ tered to promote the entymatic conversion of CN to thiocyanate, which is readily processed by the body and excreted in urine.2'1· 10 This

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4. TITLE AND SUBTITLE Noninvasive in vivo monitoring of cyanide toxicity and treatment usingdiffuse optical spectroscopy in a rabbit model

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6. AUTHOR(S) Lee J., Keuter K. A., Kim J., Tran A., Uppal A., Mukai D., Mahon S. B.,Cancio L. C., Batchinsky A., Tromberg B. J., Brenner M.,

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Noninra1irc In ~lro Monitorill l{ ofC\ {(1.\U in· Trearmull

treatment method carnes considerable mb mcludlllg -.ub­optimal nitrite dosing. or alternatively. overdosage leading to excessive MctHb, decreased oxygen carrying capacity because of Mctllb formation (particularly when combmcd with carbon monoxide toxicity), profound vasodilatiOn anu hypotension. and methylene blue-related complications .. , Although a newer. les-. tox1c treatment U\mg hydroxocobalamin ha'> been recently appro,ed by the Food and Drug Aumim.,tratlon. 1

- the limued ..,helf life. mk of anaphylaxis. reqUJrement for high­volume intravenous administration. and extremely high co .. ts of replacing all cyanide treatment kits throughout the U.S. ha<, slowed wide~pread u-.c. Although hydroxocohalamm has a high bmding affinity for C . and once bound. form-. the non­toxic cyamx:obalamm ( Vuamm B 12) MetHb mduction v. ill ~till be ,.,.idely available for future C tox1c1ty e\enh. 1

· 1 ~~

Ncar infrared diffuse optical spectroscopy (DOS) is a nonin­vasive ortlcal technology with the ability to simultancOU'>Iy determine tissue scattering and absorption properties and has the potential to provide capabilities for CN tO\ICity treatment mon itonng by a\'>Cs'>mg the pathophy.,iologic changes associ­ated '" Hh cyamde toxicity and its re\er-.al. "' J TI1ese change'> 1nclude an increase in oxyhemoglobin. decrca'>e 111 deoxyhc­moglobm. and an mcrea"e in the reduced form of C) tochrome-c oxtdasc during the induction of cyaniuc toxtclly. Conver<;ely. when cyanide toxicit} is reversed wi th ~ouium nitnte. DOS has the ability to follow the decrease of oxyhemoglobin. increa ... e of deoxyhemoglobin, the cytochrome c oxidase oxi­dation. and the increa..,e of methemoglobin 111 the ti.....,ue. The ability of DOS to stmultancously mea'>ure t1s-.ue scattenng anu absorption enables accurate. quantitative determination of the concentration of NIR-absorbing solute'> 111 huiJ.. tis'>ue" in "ivo. n--1· We propose that DOS. which can noninvasively quantify tissue oxy-. met . and deoxy hcmoglohin, 1x .JI meth­ylene blue levels.4

' as well as changes in cytochrome-c oxi­dase redox states. v.oulu he a potentially 1deal technology to fulhllthe need for cyamde toxicuy treatment monlloring.

We have pre\ iously shtmn that DOS can he used to detect the physiologic events occurring during ue\clopment of C toxicity in an ammal mouel .... Therefore. 111 thi'> "tully. we extend the imestigat1ons to determine the feasibility of using a noninvasive DOS prototype uevice we designed and con­\tructed to detect the physiologic and biochemical cvenh uur­mg '>odium nitrite-Induced methemoglobm-ba..,ed treatment of CN toxicit) in a rabbll model.

MATERIAL AND METHODS

General Preparation The procedure<, involvmg live vertebrate animals were re\iewed and approved by the full} accredited University of California Irvine !UCL) ln..,titutional An1mal Care and Usc Committee (IACUC). Male ew Lealand \\bite rabbits (N = 6) (Myrtle'<, Rabbllry Inc .. Thompson Station. Tenne-..,ee) weighing 4.0 ± 0.5 kg were anestheti;cd \\ Jth an mtramu-.­cular (IM) combination of Ketamine HCI 50 mg/J..g ( Keta-

616

JeCt. PhoeniX PharmaceutJcal Inc .. St. Joseph. MIChigan) anu Xyla;ine 5 mglkg (Anaseu. Lloyed Laboratories. Shcnandoa. Iowa). using a 23-gaugc 5/R inch needle. After the IM mjcc­llon. a 21-gauge. l-inch catheter wa<, placcu in the animal'" marglllal car \ein to aummistcr intravenous (IV) anesthcsw A continuous mfusion ol 0.7 mglkg/min Ketammc anu 0.14 mg/ J..g/mlll Xyla11ne wa-, U\ed as maintenance anc-,thcllc through­out the proccuure A plane 2 (loss ot blink rcflcxc..,, hxcd pupils. regular respmllion with ventilator as ... lstance) ucpth of anesthesia wa ... maintained throughout the surgery. hy moni­toring the phy<>ical reflexes of the animal.

The animals were intubated with a 3.0-cuflcd endotracheal tube. which \\as secured by a gautc tie. anu rncchalllcall) \Cn­tllatcd (uual phase control rc-.pirator. model 12A4BH>M-5R. llanaru Apparatw •. Ch1cago. lllinoi'>) at a rc!>p1rat1on rate of 32/mlll and a tidal volume of 50 mL and a fract1on of 1n"l))fCd oxygen ( h0

2) of I 001:f. A pulse o\imeter (B1o:>. 1700 Pul-.c

Oximeter. Ohmeda, Boulder. Colorado) with a rrobc placed on the tongue was U'ied to measure saturation of hemoglohut with o:-.ygcn (SPO,). Upon completion of the exrenmcnt. the ani mab \\.Cre euthanited \\lth an mtra\enou-. lllJCLllon of l:utha-6 (65 mg/kg) administered through the marginal car' cin.

Systemic Arterial Blood Pressure, Blood Gas Analysis, and Complete Blood Counts Femoral arterial anu venou-. cutdowns were performed for central line placement to collect blood sample!<> and mea<,ure sy'>temic pressure. A 3-inch mc1sion was malic v. ith a I 0 blade scalpel blade on the \ha\ed. left hmd leg of the animal. and blunt dls-,ectlon Wa\ U'>ec.l to 1\0iatc the \ell\ anu arter). A 12 inLh <4 French). IX gauge catheter (C PMA 400 rA. Cook lnL .. Bloomington. Indiana) \\US inserted tnto each. anu a 3-way ~topcock was placed on the end<,. Systenuc arterial prcs\ure was monitorcu using a calibrated pressure transducer (TSDI04A Tran-,ducer anu MPIOO WSW Sy..,tem. B10pac Sy-.tem,, Inc .. Santa Barbara. Cahfomia). Blood pres,ure \\a\ momtored to as..,ure the stability of the .m1mal dunng the proce­dure. Blood wa!> Withdrawn from both the arterial anu \Cnou ... line .. anu mea\ured h} •• blood gas analy7cr (IRMA SL Sene\ 2000 Blood Analy'>is System, Diametrics Medical Inc .. St. Paul. Minnesota) to obtain ABG and VBG. Auuitional blood \\LI\ drav.n from the artery and was sent to an out..,1ue laclllly (Antcch Diagnostics. lr\JOC. California) lor complete blood count (CBC) analys1-. and cyamde levels of \\hole blood.

Noninvasive Measurements Using Diffuse Optical Spectroscopy (DOS) DJ!'fw,e optical ~pectroscopy mea<,urements were obtamed through a fiber-optic probe with a light diouc cmlltcr and detector at a fixed di-.tance ( 1.0 em) from the source fiber. wh1ch \\a\ placed in the -.urface of the -.haveu nght. hllld. 1nner th1gh olthe animal. The broadband DOS prototype s)stcm \\C

con-.tructcd ·~ ~· -~~ comb1ne' mu I tJfrequenc) domam photon 1mgratJon <FDPM) \\ith t1me-mdcpendent near mlrared ( IRJ spectro.,copy to accurately mca.,ure bulk ti.....,ue ab-.orptlon and

MI LIT\R\ IF-DICJNE. Vol 174. June 2009

Nonim·a1il·e In Vi\'() Moniwring of CN Toxicity Treatment

scattering spectra. The DOS prototype employs six laser d1odes at d1screte wavelengths (661. 681, 783. 823. 850, and 910 nm), and a tiber coupled avalanche photo diode detector for the fre­quency domain measuremenh. The reduced scattering coef­ficient" are calculated as a function of wavelength throughout the NIR region by fitting a power law to the<.,e '>i\ reduced ~cat­tenng coefficients. The steady-state acquisition is a broadband retlectam:e measurement from 600 to I 000 nm that follows the frequency dommn (FD) mea'ourements using a tungsten-halo­gen light source (FiberLite lamp) and a miniature spectrome­ter (Ocean Optics USB2000). The intensity of the steady-state (SS) reflectance measurement!> are calibrated to the FD values of absorption and scattering to establish the absolute reflec­tance intensity. Finally. the tissue concentrations of oxyhe­moglobin (OIIb) dcoxyhcmoglogin (RHb). MetHb, and H,O arc calculated by a linear least squares tit of the wavelength­dependent extinction coefficient spectra of each chromophore. We used Ollb. Rllb. and MetHb absorption spectra reported by Lijllstra~" for the subsequent fitting and analysis.

CN Infusion and Toxicity Reversal

A sodium CN solution of 8 mg/60 cc normal saline was infu.,ed over 40 to 60 minute~. After this wa~ completed, a sodium nitrite !>olution of 8 mg/mL was infused at a rate of 1.4 cc per minute for another 40 to 60 minutes. Both solu­tions were infu,ed through the femoral vein using an auto­mated infu~er (GENIE Plus Infusion and Withdrawal Syringe Pump. Kent Scientific, Torrington. Connecticut). During this t1me DOS mca<,urcments were tal...en continuously and venous and arterial blood gases were drawn every 15 min for oxy­gen content and CN levcb. Each set of measurements tool... an aYerage of 5 min to complete.

RESULTS Figure I show" the extinction coefficients of the mam chro­mophores in the near infrared wmelength range between 600

:o 40 A - - OHb

--RHb IS

..... .. .J '· · · · · M!!Hb 16 :liE _e 30

., - · -MB 14

e t: ~ .§. : .

10} z :o 0 F s Iii u u 1:

z ......

' ~ F >< I 0 tal 4

0~ .....

: --------00 - - .. -.. - . - .. - .. - 0

~0 ·oo soo 900 1000

\\"A\'EU:NGTH (nm)

and I 000 nm. Figure I A show~ the exttnct1on coefficients of OHb. RHb. and MetHb and Figure I B shows the e\tmc­tion coefficients of the redo\ states of cytochromc-c ox1dase. water, and lipid. '~A150·' The measurement results from broad­band DOS were compared with co-oximetr) data from the venous blood samples.

In the following sections, the concentratiOn of chro mophore!> is represented with square bracl,ets. For example. the tissue concentration of oxyhemoglobin is :-.hnwn as IOl lbl.

Figure 2 depicts the progression of Oll b. RHb. total hemoglobin (THb). and MetHb fraction during CN infu­sion and after NaN0

1 infusion quantified from the broad­

band DOS measurements. The mea<,urement' shown in thi' figure were acquired continuou<.ly every 36 seconds from a '>ingle animal. Figure 2 clearly o.,hows the 1ncrea.,e 111 OHb and decrease in RHb during C infu'>ion and the reversal dur­ing NaN0

1 infusion. The increase in q. Met llb (( MetHb I/

([OHb]+[RHb]+[MetHb))*IOOCf-) following the <,tart of the Na 0~ infusion started is seen.

From the venous gas analysi<... the partial pressures of the venom. blood were 35.1 ± 3.5 mmHg, 46.5 ± 6.2 mmHg. and 24.3 ± 2.9 mmHg at baseline. po<..t C infU'>IOn, and po't NaN0

1 infusion, respecti vely. The respective blood cyanide

levels were 0.0. 97 ± 28. and 407 ± I 07 mg/dL. Both blood analyses are indicative of cyanide toxicity.

Figure 3 details the comparisons between co-oximetry measurements from venous blood and noninvasive broad­band DOS measurement'> from 6 anunals at baseline, post CN infusion, and post NaN0

1 treatments. hgurc 3A shows

hemoglobin concentration (sHgb) by co oximetry vs. broad­band DOS THb normalited by the respective baseline values. Both co-oximetry and broadband DOS THb 'a lues decreased throughout the experiment. A similar trend 111 THb is shov.n m Figure 2. Figure 3B shows the changes 111 Ollb fract1on trom the co-oximetry and tissue oxygen saturation (S

10,l from

broadband DOS measurements. The co-oximetry values were obtained from venous blood samples, and the resulh correlated

12 B v.· •• 005

10 "'"' ' . 004 J ,' 1 CcO_lle •

I • ~ 8 s '

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6 0031 .

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0 ~

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.,

.d~~~~-oC1l 001

2 \• CcO_Os ~ \ .. <+. 000

0 - "C)(, 100 goo 1000

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FIGURE 1. [~\lmctlnl\ cnefhcoenh of mam lls\ue chrnrnnphorcs in the ncar mfrared wavelength range~ between 600 and 1000 nm ( 1\l (h~ . dc1>X}. and melhcrnoglobm cxiiiK'Iinn codficoenh. (8) The rcuox 'tales of cyw..:hromc c oxodase. water. and lopod exunclion coefficient,.

MILITARY MEDICINE, Vol. 17-1. June 2009 617

Noninmsil·e /11 Vil'O Monitorinf? of CN To.1icirr Trearmenl

.. :I .. ..

!:=

SO r---------------------------------~1 2

~ THb

10

40

8

4

20

2

20 40 60 80

TIME(min)

FIGURE 2. Changes in oxyhemoglobin, deoxyhemoglobJO, total hemo­globm. and mcrhemoglobm fraction during cyamdc JOfm.ion and afrer '1xhum nitrite infu>Jnn tJUUilllfied from the broadband DOS measuremcnrs. The measurements were ac4uired continuously every 36 seconds from a \inglc animal.

A IZZJ.u;t _c-.

!DO - TIIo_I)OS

i1 £-t! e .: ~ : - J jl' II)

q !

di)

l)

c IZZJCOOX

- DOS IS

# i iD '! j

well with p0 2 measurements from blood gas analy~i ~. Figure 3C shows the changes in% MetHb from both the co-ox imetry and the broadband DOS measurements. F igure 3D 'hows the changes in redox states of cytochrome-c oxidase at the ba'c line. immediate ly post CN infusio n and post NaNO, infu, ion. The results in Figure 3D indicate that redox -.tate-. of cyto­chrome-c oxidase shifted toward a more reduced state during the CN infusion and reversed back toward baseline values afte r sodium mtrite infusion. Even though a "gold standard" mea­surement of cytochrome-cox idase redox slates is not avai !able. the results from blood ga~ analysis, CN level tests, co-ox imetry measurements, and broadband DOS measurements are a ll con­sistent in indicating the development of cyanide toxic ity of ani­mals and the subsequent reversal o f toxicity.

Fina lly. Figure 4 shows the corre lation between co-oxime­try and broadband DOS %- MetHb values (r = 0.85).

DISCUSSION CN tox ic ity is potent ia lly lethal and can progress very rapid ly . Therefore, early and accurate diagnosis as well as determina-

fill

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FIGURE 3. Compan\on herween co-ox1metry mea~uremenl~ from the venou~ blood and nomn~a.,ive broadband DOS measuremenl~ from 6 <lllllllaf, a1

the ba.,ehne. post ly<tn Jde infus1on. and p<N sodium nilrite <NaNO,) trearmenl. (A) Normahted ..:o-oxJmerry hemoglobm conccnlrallon h llgb) v .. Tllh by the baseline values. (8) Co-oximerry oxyhemoglobin tract Jon vs. broadband DOS w.~ue oxygen ,aluration tS 0 ,). (C) Percen1 mcrhcmoglobm 'alue' from co-oximetry and broadband DOS. (D) Changes 111 redox states of cyrochrome c oxidase (<'.CeO ox idued /\CeO "reduced) measured by broadband DOS. All seven variables were found stall~lically significam ( p < 0.05) by repeated measures analySJ\ of van a nee <ANOVA) tesl., ( SYSTAT vcrSJon 10. SPSS. ilK 1 Bars represem slandard error.

tion of the extent o f exposure i ~ essentia l. The only readily avai lable methods to detect CN exposure are blood or urine C N level tests.'! In addition to the previously mentioned limi­tations of blood cyanide level testing. 11 11 MetHb formation

618

may actually increase measured bl ood CN levels from relea-.c o f CN from cyanomethemoglobin during the assay proces-.. despite reversing the toxic effects_.un In contra<;t, noninvastve DOS methods are potentially capable of de tecting the physr-

MILITARY MEDICINE, Vol. 174, June 2009

Noninvasive In Vim Monitoring of CN Toxicity Treatment

16

,..... '*-'-"'

12

(/)

0 0 8 -o §

.D -o C';l

e 4 t:!l

0

o/oMetHb

, ·- ..... • -.-. ..... ...

0 4 8

Comumetry ("/o)

• .... ,

,. ~-. • 2

r =0.85

12 16

FIGURE 4. Correlation between co-o~imetry vs. broadband DOS percent methemoglobin values. r denote, Pearson·~ correlation r value.

ologic manifestations and biochemical effects of CN toxicity and treatment as seen in this study, even though blood levels of CN are not directly measured.

In this s tudy we evaluated the capabilities of DOS for determining the physiological effects resulting from infusion of CN to induce toxic ity followed by the infusion of sodium nitrite to reverse its toxic e ffects. This study was based on our previously described CN model,46 which was desig ned to study the capabilities of DOS to optically monitor the induc­tion of CN toxicity. The prior study46 showed that DOS was able to measure the severity of in vivo CN tox ic ity and result­ing phys iological changes noninvasively. In the current study, we investigated the ability to monito r lhe CN toxicity treat­ment reversal process with DOS. To validate the results. DOS measurements were compared to arterial and venous blood gases and also with hemoglobin co-oximetry (the standard method for blood sampling).

The current standard therapies for CN toxicity include e ither induction of methemoglobinemia or direct binding of CN with hydroxocobalamin. Nitrite-induced MetHb produc­tion is used in CN treatment because CN ions have a high affinity for MetHb and Hb is present in very high concen­trations in the blood. This high affinity leads to binding of CN in the extracellul ar space. which result!> in transfer of CN from cytochrome-c ox idase (though nitrites may also have additional mechanisms o f action inc luding direct effects on cytochro me-c oxidase activity).! '· 16·~~ The overall effect is the remova l of CN fro m cytochro mes and unblocking of the electron transport process. The first step in methemo­globin induct ion in this treatment invol ves the inha latio n of amyl nitrite vapor followed by intravenous administration o f sodium nitrite until the methemoglobin leve l reaches the desired I 0-20% of total hemoglobin.2 A level of MetHb of up to 20-30% total hemoglobin is usually tolerated well by healthy adults.2·26

MILITARY MEDICINE, Vol. 174. June 2009

Although this method of treatment may reduce the sever­ity of CN poi soning, it may also lead to other physiological complications associated with MetHb. Although MetHb mol­ecules have beneficial effects in binding CN, they become incapable of binding and delivering oxygen into tissues. This leads to abnormal oxygen affinity, reduced oxygen-carrying capacity, and potentially to ti ssue hypox i a. ~ As the levels of MetHb approach 30%, onset of physical complications begin to deve lop including dizziness, fatigue, and shortness of breath .~ Consequently, induction of MetHb may be contrain­dicated in patients with concurrent carboxyhemoglobincmia (a frequent occurrence with exposure to fires and combustion of CN-forming materials), because of further decreased oxy­gen carrying capac ity of the blood.~

Thus. induc tion of MetHb for treatment o f CN toxicity carries ri sks, and methods for careful monitoring of me th­emoglobin induc tion and effects could be of substantia l c linical benefit in CN treatment as well as MetHb toxicity. Furthermore, accurate detection of the appearance of MctHb during nitrite administration could potentially be useful as a guide for continued therapy because MetHb binds cyanide to form cyano-MetHb (which is not detected as MetHb).~n Thus rising MetHb concentrations suggest that little or no free cya­nide remains. Tn this study, we show the ability of DOS to accurately measure MetHb formation and concentration pro­viding a potential method for improving the safety and preci­sion of the MetHb induction process.

CeO is a terminal oxidase in aerobic respiration. This enzyme is involved in >95% of the oxygen consumption in the body and is essential for the e ffic ient gene ration of cel­lular ATP.51 Four redox active metal centers are present in CeO: two hemes (Cyt a and Cyt a3) and two coppers (CuA and CuB). Cyt a and C uA mediate e lectron transfer from cytochrome c to the oxygen reduction site that contains Cyt a3, and CuB . Cyt a3 is a site for binding respiratory inhibi­tors including CN. carbon dioxide, nitric oxide, nitrate, and oxygen. The ir metal centers give rise to absorption bands in the near infrared region . ~~ Especiall y in the NlR region between 600 and I 000 nm. the oxidized states have broad absorptions centered at 830 nm, which are associated with the CuA center. As these four meta l cente rs undergo changes in redox states, they give rise to changes in absorpt ion spec­tra. Since the spectral contribution of CuB associated with Cyt a3 is reported to be low (i n the range of 0- 15% ).l 1

DOS can be used to follow the redox changes of CeO from absorption band changes because of C uA and Cyt a. As cyanide binds to Cyt a3 of CeO, it prevents oxygen reduc­tio n by e lectrons leaving CuA and Cyt a. As a result. both of these NIR o ptica lly predominant metal cente rs become reduced .22 24 The reduction of these meta l centers and bind­ing of CN ligands to CeO leads to the appearance o f a s trong absorption band at 605 nmH!~ and concomitant absence of absorption bands at 655 nm and 830 nm.~~ DOS can detect the changes observed in the near infrared absorption spectra of cytochrome-c oxidase.

619

Nonim·a1irt In ~1m Mo111toring of' C\ l o.ril'irv Trcatml'llf

Re..,ult' from our previous studies and this current mvestl­gat ton demon..,trate the progressi\e reductton ol C)tochrome c oxtdase dunng C toxtctt} observed by DOS. These change.., arc concurrent \\lth the increase in mixed Yenou" nx}genatton mca..,ured hy co-oxtmctf) and the increases in tt\\Ue hemo­glohm -.aturat ton measured by DOS. During reversal of C toxicity with the formation of MetHb, the c.:ytochromc-c oxi­dase reductton is reversed in parallel with a decrea~e in mixed vcnou ... oxygen content and tissue hemoglobin oxygen 'atura­tion (as ti \suc' arc again able to extract oxygen from the cir c.:ulating blood) as measured by DOS.

There is a m].. of development of toxic level-. of Metllh from cxccssi\C nitrite admini ... tration. Metllb can he rcvcr,ed with adminrstratton of methylene blue. a cofactor for ADPH methcmoglohm reductase, an alternative entyme path\\oay 111

methemoglohm reduction. In prior studies. we demonstrated the ability of DOS to measure methylene hlue concentratton' followmg intravenou-, admin i stration .J~ Continuom. monltor­mg of tt -.sue hemoglobm oxygen saturation and cytochromi!-C oxtdation \late during MetHb reversal should help to Cll'.urc again'>! recurrence of CN toxicity from premature reversal or identtfy pcr..,istent CN toxicity sources.

Gt\en this complexity of events during C toxH:ity and treatment. DOS \hould ma]..e it possible to more re liably. safely, and noninvasively mon itor the phases of CN toxicity, Mctllb !ormation. and reversal.

There an.• a number of limitations of the ~tudy. The purpm.c of this investigation was to demonstrate feasibility of w .. ing DOS tcchnol(lgtes for detection of the ph) siologic eff ects of cyantdc tO\Ici ty and monitoring response to therapy. A-, <,uch. we did not demonstrate detimuve efficacy of MetHh tre.ttment in companson to control animals. nor in compan,on to other fonm of treatment. Additionally. CN levels remamed htgh 10

the treatment ant mal~ bec.tuse we did not ~ee].. complete rever­"al of C . Be~au-,e we dtd not follow MetHb mducllon \\ ith thiosulfate. CN wa~ not rapidly excreted from the body. and therefore remained 111 the ammal bloodstream. All ammab 111

these studie~ are anesthetitcd for comfo11 and safet) assur­ances in compliance with animal welfare regulations. f:Jfcc.:ts of ancsthesra 0 11 the phy'iiologtc responses to CN poi soning and response to treatment cannot be asses-.ed.

Specific.: DOS limitations include the fact that DOS mea -,urcs average tissue constituents to a depth of apprO\nnately 2 -4 em (at the source detector <,eparation of I 0 mm used here). The current sy,tem measures only one \lie. \\-hich i" placed O\er mu'>clc. hut mcludes contribution~ from s].. in and fat lay­ers. Regional vanabthty. or '-pecific.: organ dysfunctton that arc ]..nO\\ n to occur wtth C e\posure: would not be detected with the tuneut sclllp. Future designs \\-ith collec.:uon 10 paral­lel from multtple ~ource-detector separation-. and \ite\ could reduc.:e potentral \Jriabilit} from these factors and tt\sue het­erogeneity would be better charac.:terited . In addition. \'-llhm the DOS hemoglobm signals. there may be some mterferem:e of tissue myoglobin or other NJR absorbers not accounted lor in the analy\is

620

DOS appears to prmtde a novel tool f()r futun: \llldt e~ of CN toxtcit) and re\er,al. may he helpful for \!Utile' ~.·ompar­

mg the .... tfet) and effecti\ene,., of the'e \anou' treatment reg­tmen\, and ha~ potential to be useful for a range ol dtntcal condition\ "'here Ill-\ tvo NIR ·ahsorhmg chromophore con centratlon measurement' nla) he hcnefiual.

ACKNOWLEDGMENTS

w~ !hank 1 anya Bumey for ''""liiiH:l' \ .. llh lim prujl'll l'lm \\ork Wil\ 'liP pnncd h) '\tr l·orce Scu!nllhc Rc-.earth Medtc;tll1ec l .kcln1111'1ogralll a~·rec mem no. \ 1·-95:10 04 1·0101. hy t.a-.er Mll'rnhcam ilntl Med1~al Progr<~m. Beckman I a'er ln'olltule. l lm\Cf\11} of C.tillornl.t lr,mc Inn RROIIlJ2l. and Na11nn.1l ln,lllule' of llc,tllh (no. I U:'i4NS 06.171!! 01 l .

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