ORIGINAL ARTICLE

Toluidine blue for the intraoperative staining of the ureters.Studies on the safe administration in rats

Frank Petrat & Matthias Hartmann & Ernst Schmidt &Florian Grabellus & Tim Hamburger & Herbert de Groot

Received: 10 October 2011 /Accepted: 10 January 2012 /Published online: 2 February 2012# Springer-Verlag 2012

AbstractPurpose Acute cardiovascular events have repeatedly beenreported to occur during the intraoperative presentation ofthe urinary tract with toluidine blue (TB). We here assessedthe minimum TB dose required, and its safest and mostsuitable form of intravenous administration for the intra-operative staining of the ureters in rats.Methods TB (0.13, 0.4, 1.3, or 4.0 mg/kg) was administeredto anesthetized rats either by intravenous injection within1 min or by infusion within 10 min. During the experiments,biomonitoring parameters such as electrocardiograms (ECGs)and mean arterial blood pressure (MAP) were recorded,blood gas analysis was performed, and methemoglobin

measured. Tissue injury was assessed from released plasmaenzyme activities and histopathologically. The intraopera-tive staining of the ureters was documented photographi-cally, and total urinary excretion and final urine/plasma TBconcentrations were determined.Results Parameters of blood gas analysis, methemoglobinconcentrations, and markers of tissue injury were slightlyaffected by the two highest TB doses but not at all by thelower ones. At doses of ≥0.4 mg/kg, ureters were stainedsufficiently. Staining was more intense, and urine excre-tion of TB higher on average when the dye was injected.The 1-min injection of ≥1.3 mg TB/kg strongly and tem-porarily decreased the MAP, while the infusions causedlesser effects. Mean ECG parameters were not affected byany TB administration, but one animal developed a tem-porary bundle branch block after the 1-min injection of4.0 mg/kg.Conclusions In rats, intravenous injection of 0.4 mg TB/kgwas sufficient for the intraoperative staining of the urinarytract without the risk of severe cardiovascular and hemo-dynamic side effects. Provided our results are transferableto humans, the administration of low TB doses could allowits safer clinical use for the intraoperative visualization ofthe ureters.

Keywords Cardiovascular . Hemodynamics . Toluidineblue . Urinary tract . Methemoglobin

AbbreviationsTB Toluidine blueECG ElectrocardiogramMAP Mean arterial blood pressureMB Methylene bluePO2 Arterial oxygen partial pressure

F. Petrat (*) : T. Hamburger :H. de GrootInstitut für Physiologische Chemie, Universitätsklinikum,Universität Duisburg-Essen,Hufelandstr. 55,45122 Essen, Germanye-mail: frank.petrat@uni-duisburg-essen.de

M. HartmannKlinik für Anästhesiologie und Intensivmedizin,Universitätsklinikum Essen,Hufelandstr. 55,45122 Essen, Germany

E. SchmidtDr. Franz Köhler Chemie,Werner-von-Siemens-Str. 22,64625 Bensheim, Germany

F. GrabellusInstitut für Pathologie und Neuropathologie,Universitätsklinikum Essen,Hufelandstr. 55,45122 Essen, Germany

Langenbecks Arch Surg (2012) 397:983–993DOI 10.1007/s00423-012-0907-y

metHb MethemoglobinHb HemoglobinSO2 Oxygen saturation

Introduction

Toluidine blue (TB; active ingredient: o-tolonium chloride, 3-amino-7-dimethylamino-2-methylphenazothionium chloride)is a redox-active, cationic, metachromatic thiazine dye, whichhas been used for many years as an antidote for the intrave-nous treatment of life-threatening methemoglobinemia afterpoisoning with, e.g., nitrites, chromates, nitrobenzene, 4-dimethylaminophenol, and hydrogen cyanide [1, 2]. For thispurpose, it is slowly administered intravenously at a dose of2–4 mg/kg, as recommended by the manufacturer (Dr. F.Köhler Chemie, Bensheim, Germany). In addition, since2007 TB is approved in Germany for the topical use in intra-operative vital staining of the parathyroid glands, for chro-moendoscopy, chromolaparoscopy, and to constitute fistulas(Dr. F. Köhler Chemie GmbH: Gebrauchs- und Fachinforma-tion “Toluidinblau®”. Stand: Januar 2010). In recent years, ithas increasingly been used for non-approved indications inurological and gynecological surgery, as the previously usedthiazine dyemethylene blue (MB) has lost its clinical approvalfor parenteral administration in Europe [3]. Here, TB is intra-venously administered to examine or verify the intraoperativeurine flow, e.g., to exclude stenosis of or leakage from theureters and to stain the ureters in order to decrease the risk ofintraoperative ureteral injury. Since 2007, some acute cardio-vascular events such as bradycardia, asystole, and cardiacarrest have been described to occur during the intraoperativepresentation of the urinary tract with intravenous TB, oftenrequiring resuscitation [4, 5]. In all these cases, 150–300 mg TB per patient, i.e., doses as also indicated for treatingmethemoglobinemia, was intraoperatively administered intra-venously during anesthesia, and the complications occurredwithin 10 min. For this reason, the Drug Commission of theGerman Medical Association and the manufacturer havewarned of the serious circulatory responses, which may occurduring the “off-label-use” of 300 mg TB/patient. However,due to the lack of alternative drugs, it is expected that TB willstill be used for the representation of the urinary tract.

Although the transferability to the clinical situation canonly be proposed, we here try to provide recommendationsfor the most safe off-label-use of TB based on a rat model.For this, we assessed (1) the minimum required TB dose forthe intraoperative presentation of the ureters and (2) itsdependence on the rate of administration, and (3) identifiedeffects on the cardiovascular system, other vital/systemicparameters, and markers of tissue injury as a function ofthe dose and rate of administration.

Materials and methods

Chemicals/materials

TB (30 mg/ml) was provided by Dr. F. Köhler Chemie(Bensheim, Germany). Potassium ferricyanide and potassi-um cyanide were from Merck (Darmstadt, Germany). For-malin solution (10%, buffered) was obtained from Fluka/Sigma-Aldrich (Steinheim, Germany), isoflurane (Florene)from Abbott (Wiesbaden, Germany), ketamine 10% fromCeva (Düsseldorf, Germany), lidocaine (Xylocain 1%) fromAstraZeneca (Wedel, Germany), and Ringer’s solutionMacoflex N from MacoPharma International (Langen,Germany). Portex catheters (0.58 mm inner diameter,0.96 mm outer diameter) were purchased from Smiths Med-ical International (Hythe, UK). Paraffin (Paraplast TissueEmbedding Medium REF 501006) was from McCormickScientific (St. Louis, MO, USA), and medical oxygen fromAir Liquide (Düsseldorf, Germany).

Animals

A total number of 60 male Wistar rats (Rattus norvegicus,424±23 g; 400–470 g; 10–14 weeks old) were obtained fromthe central animal unit of the Essen University Hospital.Animals were kept under standardized conditions of temper-ature (22±1°C), humidity (55±5%), and 12/12-h light/darkcycles with free access to food (ssniff-Spezialdiäten, Soest,Germany) and water. All animals received humane careaccording to the standards of Annex III of the directive2010/63/EU of the European Parliament and of the Councilof 22 September 2010 on the protection of animals used forscientific purposes [6]. The experimental protocol was ap-proved by the local committee based on the local animalprotection act.

Anesthesia, analgesia, and surgical procedure

Rats were anesthetized with isoflurane (1.0–1.5% in 100%medical O2 at 1.0 l/min) through face masks connected to avaporizer and received ketamine (50 mg/kg body weight s.c.) for analgesia. After local lidocaine administration (5 mg/kg s.c.), a median skin-deep inguinal incision of about 2 cmwas made, and a Portex catheter (0.58 mm ID, 0.96 mmOD) was placed within the right femoral artery and the rightfemoral vein. Thereafter, a median abdominal laparotomy ofabout 5 cm was performed along the linea alba, with thesmall intestine carefully mobilized out of the abdominalcavity and placed on moistened compresses. Through asmall incision in the urinary bladder wall, a Portex catheterwas introduced into the bladder and fixed with surgicalsuture. To allow a continuous visual observation of theureters and to minimize evaporation, the wound edges were

984 Langenbecks Arch Surg (2012) 397:983–993

spread with retractors, the abdominal cavity was coveredwith cling film, and the area of operation illuminated withoperating light. At the end of experiment, animals weresacrificed by resection of the heart (and other organs) underdeep isoflurane anesthesia.

Administration of TB and experimental groups

TB was administered intravenously with the rate of admin-istration based on the clinical practice, i.e., short injection orshort infusion. Doses were converted to rats based on a bodyweight of 75 kg/human. The experiments were performedwith six rats per group to compare the effects of 10, 30, 100,and 300 mg TB/75 kg, i.e., 0.13, 0.40, 1.33, and 4.0 mg TB/kg. The doses were administered via the V. femoralis, eitheras an injection of 0.5 ml/kg within 1 min (followed by0.5 ml saline to flush the TB out of the catheter) or as aninfusion of 1.33 ml/kg (i.e., 100 ml/75 kg) over 10 min(Perfusor-Secura FT; B. BRAUN, Melsungen, Germany).The dye was protected from light before and during itsadministration. To obtain the respective doses and volumes,aliquots of the TB stock solution (30 mg/ml) were freshlydiluted with sterile 0.9% NaCl. Outside the TB administra-tion periods, 0.9% NaCl solution was administered (7 ml/kg × h). An injection and an infusion control group received0.9% NaCl without TB. The following experimental groupswere compared:

– Injection control group (0.5 ml 0.9% NaCl/kg within1 min, no TB)

– Infusion control group (1.33 ml 0.9% NaCl/kg within10 min, no TB)

– 0.13-mg TB injection group (0.13 mg TB/kg within1 min)

– 0.13-mg TB infusion group (0.13 mg TB/kg within10 min)

– 0.40-mg TB injection group (0.40 mg TB/kg within1 min)

– 0.40-mg TB infusion group (0.40 mg TB/kg within10 min)

– 1.33-mg TB injection group (1.33 mg TB/kg within1 min)

– 1.33-mg TB infusion group (1.33 mg TB/kg within10 min)

– 4.0-mg TB injection group (4.0 mg TB/kg within 1 min)– 4.0-mg TB infusion group (4.0 mg TB/kg kg within

10 min)

Biomonitoring

Electrocardiograms (ECGs) were derived continuously us-ing a three-lead configuration, recorded at a sampling rateof 40 kHz (PowerLab 26T; ADInstruments, Spechbach,

Germany), and displayed on a monitor (Sirecust 1281;Siemens, Essen, Germany) using the LabChart 7 Pro soft-ware (ADInstruments, Spechbach, Germany). The ECGparameters recommended by A. K. Farraj et al. for smallrodents in toxicological studies [7] were manually assessedin a blinded fashion and evaluated for the time points givenin “Results” section.

Systolic, diastolic, and mean arterial blood pressures(MAP) were continuously recorded via the femoral arterycatheter that was connected with a pressure transducer (MX960; Medex Medical, Rossendale, UK) and displayed on themonitor. Ringer’s solution was infused at a rate of 3 ml/h tokeep the catheter functional. Heart rates were determinedfrom systolic blood pressure spikes. The rectal temperaturewas continuously monitored using a rectal sensor and main-tained at 37±0.5°C by an underlying thermostated operatingtable and a heat lamp above the area of operations. Theoxygen saturation was recorded using a pulse oximeter(OxiCliq A; Nellcor, Boulder, CO, USA) placed at the righthind limb. The breathing rate was determined based on theventilation movements in 10-min intervals.

Assessment of blood, plasma, and serum parameters

Using a 2-ml syringe (Pico50, Radiometer Medical ApS,Brønshøj, Denmark) containing 80 I.U. electrolyte-balancedheparin, blood samples (0.7 ml) were taken from the femoralartery immediately after catheter insertion, before the start ofthe TB administration, and 10, 30, and 60 min after startingthe TB injections/infusions. At the end of the experiments,blood plasma for the determination of the plasma TB,nitrite, and nitrate concentrations was obtained. For eachblood sampling, animals were substituted with 0.7 ml 0.9%NaCl solution. Arterial oxygen and carbon dioxide partialpressures (PO2, PCO2), oxygen saturation (SO2), pH, acid–base status, hemoglobin concentration, hematocrit, electro-lytes (Na+, K+, Cl−, Ca2+), metabolic parameters (lactate,glucose), and osmolality were assessed with a blood gasanalyzer (ABL 715; Radiometer, Copenhagen, Denmark).Blood plasma was obtained by centrifugation at 3,000g for15 min (25°C). The plasma activities of lactate dehydro-genase, aspartate aminotransferase, and alanine amino-transferase and the plasma creatinine concentration weredetermined with a fully automated clinical chemistry ana-lyzer (Vitalab Selectra E; VWR International, Darmstadt,Germany).

Immediately after each blood sampling, the percentage ofmethemoglobin (metHb) from the total hemoglobin (Hb)was determined according to [8], with slight modifications.Briefly, 50 μl of blood per sample was mixed with 1.25 mlof A. bidest. and incubated for 10 min. Then, 1.25-mlHEPES buffer (50 mM, pH 7.0) was added and the samplecentrifuged (10,000g for 10 min at 25°C). One milliliter of

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the supernatant was supplemented with (A) 10 μl of potas-sium ferricyanide (152 mM), and another one with (B) 10 μlof A. bidest. The absorption of both supernatants was deter-mined at 630 nm 1 min later (E1(A), E1(B); blank: A.bidest.). Afterwards, 10 μl of potassium cyanide (77 mM)was added to both supernatants and 5 min later their absorp-tions were determined again (E2(A), E2(B)). The percentageof metHb from the total Hb was finally calculated by

metHb %ð Þ ¼ ½E1 Bð Þ � E2 Bð Þ� � 100

E1 Að Þ � E2 Að Þ :

TB concentrations in the final plasma at the end of theexperiments were determined from the TB absorption max-imum at 636 nm and calibration curves obtained from TBstandards under the same settings. For this purpose, plasmasamples were mixed 1:1 with 0.9% NaCl solution andspectra recorded (350–750 nm).

Nitrite and nitrate (as a measure of nitric oxide) weredetermined by the Griess method [9], using a commerciallyavailable photometric nitrate test (R-Biopharm, Darmstadt,Germany), with some modifications. Final plasma sampleswere filtered (Vivaspin 500, 30,000 MW, Sartorius StedimBiotech, Göttingen, Germany) by centrifugation (16,000g,30 min, 4°C) subsequent to a wash step of the filters with A.bidest. to remove possible contaminating nitrate. Samples(100 μl) of the plasma filtrate were mixed with phosphatebuffer (100 μl, 50 mM, pH 7.4), then 100 μl of a NADPH/FAD solution and 5 μl of nitrate reductase solution (bothfrom the nitrate test kit of R-Biopharm) were added, and themixture incubated (20 min, 25°C). Afterwards, 5 μl ofsodium pyruvate solution (12 mg/ml A. bidest.) and 5 μllactate dehydrogenase solution (Roche, Mannheim, Ger-many, diluted 1:10 with A. bidest.) were added. Subsequentto a further incubation (20 min, 25°C), Griess reagent(750 μl; 1% sulphanilamide+0.1% N-(1-naphthyl) ethyl-enediamine dihydrochloride, 1:1) was added, then the mix-ture incubated again (10 min, 25°C), and finally theabsorption was determined at 542 nm; samples with phos-phate buffer instead of plasma served as blanks. Concen-trations were assessed from calibration curves performedunder the same settings.

Histological evaluation of the heart, lung, liver, and kidneys

At the end of the experiments, the organs were harvested,added to the 20-fold volume of formalin (10%, neutralbuffered) and fixed for at least 24 h. Before the thorax wasopened, the lung was filled with 10 cm3 air via a cannulaafter tracheotomy to avoid blood aspiration due to pneumo-thorax, then harvested, filled with formalin (10 ml) to ensurecomplete unfolding, and submersed in the fixative. Paraffin-embedded sections (2 μm each) were stained with

hematoxylin–eosin. Histopathological changes were evalu-ated in a blinded fashion using light microscopy.

The lung and the heart were scored for the mean numberof granulocytes per field of vision (ten fields of vision persection, ×400 magnification) and the mean percentage areawith edema per field of vision (20 fields of vision per section,×100 magnification). Liver sections were scored as follows:mean number of erythrocytes in the sinusoids per field ofvision (15 fields of vision per section, ×1000 magnification),mean percentage area of granulocyte infiltrates in the liverparenchyma per field of vision (20 fields of vision per sec-tion, ×40 magnification), mean percentage of liver vein bloodfilling states (bloodless/blood-filled/thrombi; whole section,×40 magnification), and the mean percentage number andlocation (close to vessels, within parenchyma) of hepatocyteswith cytosolic vacuoles (small, big, mixed) per field of vision(15 fields of vision per section, ×400 magnification). Kidneysections were graded according to: percentage area of theinner and outer renal medulla and of the cortex containingerythrocytes (×40 magnification), percentage number of atro-phic glomeruli (×100 magnification), and of glomeruli withmore than 20 erythrocytes (×400 magnification) of the wholesection, and percentage area of the inner and outer renalmedulla and of the cortex containing tubuli with detachedbasement membranes (×400 magnification).

Documentation of the ureter staining with TBand determination of the total urinary excretionand TB urine concentration

The intraoperative staining of the ureters following intrave-nous TB injections or infusions was documented photo-graphically 10, 30, and 60 min after TB administration. Inaddition, the time between the end of the administration andthe first visual detectability of TB within the ureters wasnoted to assess the kinetics of ureter staining.

At the end of the experiments, the total volume of urine,collected by means of the urinary catheter, and the TBconcentration in the urine were determined. Urine TB con-centrations were assessed from the TB absorption maximumat 636 nm based on calibrations performed with TB stand-ards under the same settings.

Statistics

Experiments were performed with six animals per experi-mental group. Biochemical assays were run in duplicateunless stated otherwise. The data are expressed as meanvalues ± SEM. Comparisons among multiple groups wereperformed using either one-way analysis of variance(ANOVA) for nonrecurring or for repeated measures ortwo-way ANOVA followed by the Fisher (LSD) post hocanalysis. A p value of <0.05 was considered significant.

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Results

Effect of TB on MAP, ECG, and other vital parameters

The MAP of all animals was around 100 mmHg when theexperimental procedure started (Fig. 1). In the injectionand the infusion control animals (i.e., no TB administra-tion), it remained at this value during the whole experi-ment. When the different TB doses were intravenouslyinjected during 1 min, the MAP abruptly dropped in adose-dependent manner (Fig. 1a). In animals receiving4.0 mg TB/kg, it decreased to a mean value of 56.1±9.4 mmHg and to individual values as low as 26 and44 mmHg in two animals (range 26.0–90.5 mmHg). Sub-sequent to the decrease in MAP, it gradually normalized tovalues slightly below the MAP of the injection controlgroup. Less abrupt and severe hemodynamic effectsresulted from the 10-min infusions of the same TB doses(Fig. 1b). MAP values were the lowest 10 min after theend of the infusions (73.8±7.2 mmHg in the 4.0-mginfusion group; range 56.5–95.5 mmHg). Unlike for theTB injection groups, the effects of TB were not dose-dependent and during the whole experimental period onlythe MAP of the 4.0-mg TB infusion group was signifi-cantly lower than the one of the infusion control group.

Both, the injection or the infusion of the highest TB dosehad no significant effect on the mean ECG parametersobtained from six rats/group (Fig. 2). In a preliminary series,however, one animal died due to apnea in our spontaneousbreathing anesthesia model (data not shown). In the presentseries, in another animal, the 1-min injection of this doseresulted in a strong temporary decrease of the R amplitudelasting 7 min, which can be best explained by a bundlebranch block (Fig. 3). The individual ECGs of all the otheranimals were not affected by TB, regardless of whether thedye was injected or infused (not shown).

The heart rates of the animals receiving the highest TBdose as an injection decreased insignificantly from 288±11beats/min (range 264–336 beats/min) to 264±18 beats/min(range 216–336 beats/min) within 10 min, i.e., slower thanthe MAP, and remained around a mean value of 260 beats/min for the whole experiment (not shown). The injection ofthe lower TB doses even had smaller effects on the heart rateand there was no dose dependence (1.33-mg TB injectiongroup 8%; 0.4-mg TB injection group 1.5%; 0.13-mg TBinjection group 5%). No distinct decrease in mean heart rateoccurred when the TB doses were infused over 10 min (datanot shown).

The breathing rate and the rectal temperature were notsignificantly affected by any TB dose at any kind of admin-istration (data not shown). At the end of the 10-min infusionof 4.0 mg TB/kg, the mean SO2 as obtained by pulseoximetry temporarily dropped from 99.2±0.3% to 95.2±

1.4% (p<0.05), due to the decrease in SO2 to 91.0% intwo of the six animals, and then normalized again within10 min (not shown).

Effect of TB on parameters of blood gas analysis,electrolytes, metabolic parameters, osmolality, Hb, MetHb,and plasma nitrite/nitrate

Compared to the values obtained from the blood samplingimmediately before TB administration, the redox dye at itshigher doses mediated small and mostly temporary changesin the blood gas analyzer-derived Hb concentration and PO2,as well as in the MetHb concentration (Table 1). Thesechanges did not clearly depend on the kind of administration,i.e., injection vs. infusion, and did not occur at the lowerdoses (not shown). The blood pH, SO2, base excess, electro-lytes (Na+, K+, Ca2+, Cl−), glucose and lactate concentra-tions, osmolality, and plasma nitrite/nitrate concentrations

Fig. 1 Effects of toluidine blue injections and infusions on the meanarterial blood pressure. Mean arterial blood pressure (MAP) of anes-thetized rats was recorded via the femoral artery catheter that wasconnected with a pressure transducer. Toluidine blue (TB; 0.13, 0.4,1.33 or 4.0 mg/kg) diluted with 0.9% NaCl solution was administeredeither by (a) intravenous injection (0.5 ml/kg) within 1 min or (b) byinfusion (1.33 ml/kg) within 10 min. Control animals received thesame volumes of 0.9% NaCl solution without TB. Values shownrepresent means±SEM, n06. *p<0.05 (vs. control (period followingTB administration) for time-matched data)

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(as a measure of nitric oxide) were not significantly alteredby any TB dose (data not shown).

Effect of TB on parameters of tissue injury

In the injection and the infusion control groups, the plasmacreatinine concentration and the plasma enzyme activitiesof lactate dehydrogenase, aspartate aminotransferase, andalanine aminotransferase did not significantly change dur-ing the experimental procedure (Table 2; shown for creat-inine and lactate dehydrogenase only). Both the injectionand the infusion of 4.0 mg TB/kg slightly increased theplasma creatinine concentration and the lactate dehydroge-nase activity but not the activities of the transaminases.

The lower TB doses did not affect any of these parameters(data not shown).

The histological evaluation of the kidneys at the end ofthe experiment revealed a modest TB-dependent enrichmentof erythrocytes in the inner and outer renal medulla, thecortex, and in the glomeruli (not shown). A few atrophicglomeruli were found in some TB groups but no detachmentof the tubuli basement membrane. These mild histophatho-logical changes did not clearly depend on the TB dosesadministered. Compared to the injection control group andthe TB infusion groups, there was an insignificant tendencyof more pronounced changes to the histologic architectureof the kidneys in the 1-min injection groups. Similar to thekidney, also the liver histology was hardly affected by any

Fig. 2 Effects of toluidine blueinjections and infusions onelectrocardiographicparameters. Electrocardiogramsof anesthetized rats werederived at a three-lead configu-ration, recorded at a samplingrate of 40 kHz, and manuallyevaluated for the time pointsgiven. Toluidine blue (TB;4.0 mg/kg) diluted with 0.9%NaCl solution was administeredeither by intravenous injection(0.5 ml/kg) within 1 min or byinfusion (1.33 ml/kg) within10 min. Arrows indicate thestart of TB administrations.Values shown representmeans±SEM, n06.

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TB treatment. A slight and not clearly dose-dependent en-richment of erythrocytes was found in the sinusoids with thetendency of more erythrocytes per tissue area in the TBinfusion groups (not shown). No TB dose had an effect onthe number of vacuolized hepatocytes and on the percentagetissue areas containing inflammatory infiltrates. The histo-pathologic evaluation and comparison of the hearts and thelungs revealed no TB-dependent changes.

Effect of the TB dose and kind of administration on kineticsand intensity of the ureter staining, TB urine concentration,and total urinary excretion

The ureters of rats that received no TB were difficult todistinguish with the eye from the surrounding fatty tissue,due to their small diameter (approx. 1 mm) and pale color(Fig. 4a). The administration of 0.13 mg TB/kg did notsufficiently stain the ureters both after intravenous injectionand infusion of the dye, but peristaltic waves could beobserved within the ureters, transporting fractions of theurine/TB mixture to the urinary bladder (not shown). Atdoses of 0.4 mg TB/kg and above, ureters took on a greenishblue color (due to the mixture of urine and TB), which couldbe well differentiated from the surrounding tissue color thatremained unchanged subsequent to the 0.4-mg dose or

turned to violet-blue at 1.33 and 4.0 mg/kg (Fig. 4b–d).Ureters were stained throughout more intensive by TB whenthe dye was administered as an 1-min injection, as comparedwith the respective 10-min infusions. The time until TBbecame visible within the ureters strongly depended on itsdose and the kind of administration (Fig. 5a). At the lowerTB doses (0.13 and 0.4 mg/kg), the first occurrence of thedye within the ureters and ureter staining, respectively, tooksignificantly longer, when TB was infused. While the ure-ters were stained faster when increasing the dose infused,the injected TB required longer to stain the ureters withincreasing doses. At a TB dose of 1.33 mg/kg, the kineticsof ureter staining did not depend on the rate of administra-tion. Ureters stained with ≥0.4 mg TB could be identifiedsuccessfully until the end of the experiments, i.e., for 60 minsubsequent to TB administration.

Fig. 3 Effect of a 4.0-mg toluidine blue injection on an individual ratelectrocardiogram. The electrocardiogram of an anesthetized rat wasderived at a three-lead configuration and recorded at a sampling rate of40 kHz. Toluidine blue (TB; 4.0 mg/kg) diluted with 0.9% NaClsolution was administered by intravenous injection (0.5 ml/kg) within1 min. The electrocardiogram immediately before (a) and 3 min after(b) the TB administration is shown

Table 1 Effect of toluidine blue on PO2, Hb, and MetHb

Parameter/group Before TB 10 minafter TB

End ofexperiment

PO2 (mmHg)

Injection control 353±30 334±22 348±29

4.0 mg injection 346±16 299±13 376±22

1.33 mg injection 381±28 349±23 387±24

Infusion control 387±24 366±22 372±34

4.0 mg infusion 357±14 296±20* 369±22

1.33 mg infusion 332±25 298±25 327±32

Hb (g/dl)

Injection control 13.7±0.5 13.6±0.3 13.5±0.3

4.0 mg injection 14.0±0.2 12.7±0.2* 13.1±0.2*

1.33 mg injection 14.0±0.2 13.0±0.3* 12.8±0.3*

Infusion control 13.7±0.2 13.5±0.2 12.9±0.3

4.0 mg infusion 13.8±0.2 12.9±0.3 12.4±0.7*

1.33 mg infusion 13.2±0.2 12.5±0.4 12.1±0.2*

MetHb (%)

Injection control 0.55±0.14 0.53±0.15 0.50±0.14

4.0 mg injection 0.70±0.01 0.96±0.18 0.62±0.11

1.33 mg injection 0.45±0.07 1.21±0.24* 0.71±0.19

0.4 mg injection 0.52±0.06 0.77±0.07* 0.74±0.07*

Infusion control 0.71±0.13 0.66±0.17 0.83±0.17

4.0 mg infusion 0.64±0.10 1.26±0.18* 0.77±0.23

1.33 mg infusion 0.67±0.17 0.99±0.30 0.67±0.14

Toluidine blue (TB; 0.4, 1.33 or 4.0 mg/kg) diluted with 0.9% NaClsolution was administered either by intravenous injection (0.5 ml/kg)within 1 min or by infusion (1.33 ml/kg) within 10 min. Controlanimals received the same volumes of 0.9% NaCl solution withoutTB. Values were assessed from blood obtained immediately before and10 min after the start of TB administration, as well as from final bloodsamples. Values shown represent means±SEM, n06.

PO2 oxygen partial pressure, Hb hemoglobin, MetHb methemoglobin

*p<0.05 (vs. respective values before TB administration)

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The TB concentration within the excreted urine at the endof the experiments depended on the dose administered andwhether the dye was injected or infused (Fig. 5b). When TBwas injected within 1 min, higher doses resulted in higherurine concentrations but there was no linear relationship. Inthe TB infusion groups, the urine concentration did notfurther increase with the dose above 0.4 mg TB/kg.

Compared to the 1.33- and 4.0-mg infusion groups, signifi-cant higher TB concentrations were found in the urine, whenthe respective doses were injected, while there was no differ-ence between the 0.13- and the 0.4-mg doses, respectively. Atthe end of the experiments, no TB was detectable in bloodplasma.

The animals of the injection control group excreted atotal volume of 2.1±0.4 ml urine/kg during the whole ex-periment. In the 4.0-mg TB injection group, the total urinaryexcretion was lower (1.3±0.2 ml/kg), but not significantly.The other TB doses, regardless of whether injected or in-fused, had no effect on urine excretion, as compared to theircontrols (not shown).

Discussion

The current study demonstrates that an intravenous injectionof 0.4 mg TB/kg, i.e, a dose of 30 mg/75 kg human bodyweight, is sufficient to stain the ureters in rats without therisk of severe cardiovascular and hemodynamic side effects.

Cardiovascular and hemodynamic side effects of TB

Clinical cardiovascular complications resulting from intra-venous TB administration have been reported for decades,however, unlike for its off-label use [4, 5], only rarely [3,10]. Malignant ECG effects may be more often inducedduring the intraoperative off-label-use because anesthesiahas a number of effects on the cardiovascular system as

Table 2 Effect of toluidine blue on the plasma creatinine concentra-tion and lactate dehydrogenase activity

Parameter/group Before TB 10 minafter TB

End ofexperiment

Creatinine (mg/dl)

Injection control 0.61±0.03 0.60±0.02 0.64±0.04

4.0 mg injection 0.56±0.01 0.64±0.02* 0.67±0.01*

Infusion control 0.61±0.05 0.63±0.05 0.73±0.09

4.0 mg infusion 0.60±0.04 0.69±0.03 0.76±0.02*

Lactate dehydrogenase (U/l)

Injection control 94.3±20.3 93.6±11.8 97.0±8.0

4.0 mg injection 73.6±13.2 79.1±11.3 119.3±13.4

Infusion control 64.0±5.8 69.4±6.2 69.2±9.2

4.0 mg infusion 68.0±10.2 78.8±16.0 115.6±18.2*

Toluidine blue (TB; 4.0mg/kg) diluted with 0.9% NaCl solution wasadministered either by intravenous injection (0.5 ml/kg) within 1 minor by infusion (1.33 ml/kg) within 10 min. Control animals receivedthe same volumes of 0.9% NaCl solution without TB. Values wereassessed from blood plasma obtained immediately before and 10 minafter the start of TB administration, as well as from final blood plasma.Values shown represent means±SEM, n06.

*p<0.05 (vs. respective values before TB administration)

Fig. 4 Effect of the dose oftoluidine blue on the ureterstaining intensity. Toluidineblue (TB; 0.4, 1.33 or 4.0 mg/kg) diluted with 0.9% NaClsolution was administered byintravenous injection (0.5 ml/kg) within 1 min. Controlanimals (a) received the samevolume of 0.9% NaCl solutionwithout TB. The intraoperativestaining of the ureters wasdocumented photographically.Black arrows point on theureters. In (d) the white arrowindicates the urinary bladdercatheter containing TB. In thisanimal the left ureter wasdissected free; the right ureterwas not stained as the rightkidney excreted no TB.Photographs are representativefor six animals per group. Barsindicate 1 cm

990 Langenbecks Arch Surg (2012) 397:983–993

well. For example, general anesthesia can alter autonomictone leading to vasodilatation and eventual hypotension,while inhalation anesthetics have been shown to have pro-or antiarrhythmic effects and inhibit critical Ca2+ channelsin the heart [7, 11]. Further, the patients involved had anextensive internal medicine medical history, often includingcardiovascular diseases. In this context, it is noteworthy thatthe arrythmogene potential of TB may be enhanced byhypokalemia and hypomagnesemia, especially at prolongedQT intervals, which are associated with serious cardiacarrhythmias including torsade de pointe that can lead topotentially lethal ventricular fibrillation [7, 12, 13]. It shouldbe further noted that renal insufficiency may result in

stronger side effects, as urinary excretion of TB is impaired.Moreover, also differences in the route and rate of adminis-tration may account for the more severe intraoperative sideeffects. Heintz et al. postulated that the occurrence ofarrhythmias by TB is associated with the rate of influx inthe myocardium, and thus advised against a central venousadministration [10].

In animal studies, the effects of intravenous TB on car-diovascular and vital parameters are obviously dependent onthe species as well as on the experimental conditions, i.e.,the dose, location of the catheter, administration rate, andanesthesia/analgesia, in line with the clinical situation. Theresults of the present study indicate a high variance inindividual tolerance regarding changes in blood pressureand ECG after TB administration in rats. Already a doseof 1.33 mg/kg (100 mg TB/75 kg) bears the risk of circula-tory collapse when injected within 1 min (Fig. 1a), andserious cardiac arrhythmias may occur at 4.0 mg/kg(300 mg/75 kg; Fig. 3). Thus, a faster administration and/or higher dose probably lead to even more severe hemody-namic and cardiovascular side effects. In a study on theacute (LD50) and chronic toxicity of TB, its intravenousadministration to rats, mice, and rabbits resulted in an in-creased rate and depth of respiration, tonic convulsions,partial heart block (in many animals), and death due tocardiac and respiratory failure; symptoms of acute toxicitywere similar in all species [14]. In dogs, a 10-min infusionof 10 mg TB/kg was followed by a strongly increasedheart rate, an increase in cardiac output, and a decrease instroke volume and total peripheral resistance [15]. Arte-rial blood pressure did not change upon TB administra-tion. In these experiments TB had antiarrhythmic effectson two animals with permanent atrial flutter and elevatedventricular end-diastolic pressure, respectively. In rabbits,intravenous TB of up to 7.5 mg/kg increased the bloodpressure in a concentration-dependent manner [16]. Deathby respiratory failure was seen after administration of10 mg/kg. In comparative studies with dogs, the authorsobserved a sharp rise in blood pressure at intravenousdoses of 0.5–10 mg TB/kg. TB doses at ≥1 mg/kg pro-duced a definite bradycardia, and at ≥2.5 mg/kg, an in-crease in the height of the T wave and a high take off ofthe P wave was observed. Species-dependent reasons forthe different ECG and hemodynamic effects of TB may berelated to differences in cardiac ion channel expression [7],in vivo distribution [17], and to differences in tolerance(LD50 values) [14, 18].

The molecular pathomechanism responsible for the car-diovascular and hemodynamic side effects described hasnot been elucidated yet. TB had only minor effects onorgan histology and other parameters of tissue injury(Table 2), indicating that it affected functional/regulatoryproperties of the cardiovascular and hemodynamic system.

Fig. 5 Effect of the toluidine blue dose and kind of administration onthe kinetics of ureter staining and the final urine concentration of thedye. Toluidine blue (TB; 0.13, 0.4, 1.33 or 4.0 mg/kg) diluted with0.9% NaCl solution was administered either by intravenous injection(0.5 ml/kg) within 1 min or by infusion (1.33 ml/kg) within 10 min. In(a) the time between the end of the TB administration and the firstvisual detectability of the dye within the ureters is shown as a functionof the dose. The final concentrations of TB within the urine collectedby means of an urinary catheter is shown in (b). Values shown repre-sent means±SEM, n06. *p<0.05 (vs. respective TB infusion groups);**p<0.05 (vs. 0.13- and 0.4-mg TB injection and 0.13-mg TB infusiongroup, respectively)

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Our results do not suggest involvement of vagus activa-tion (heart rate decreased only slightly and also slowerthan the MAP), temporary alterations in (myocardial) Ca2+

homeostasis, as well as formation of MetHb (Table 1) andnitric oxide. Further, there are no indications that TBgenerally affected ventricular excitation or that it causedmyocardial ischemia, which could explain a decrease inblood pressure. Based on a circulating blood volume of60–65 ml/kg rat, a rapid injection of 4 mg TB/kg rat(300 mg TB/75 kg) would lead to a temporary bloodconcentration of about 200 μM (molecular weightTB305.83 Da). Side effects of this or even higher TB dosesmay arise from oxidation of responsible, yet unknownreductants in the endothelial cytosol and thus an imbal-ance in redox state, as well as from oxygen-dependentsuperoxide formation (redox cycling) of the dye.

Intraoperative ureter staining with TB

Upon intravenous administration, a part of TB penetratesthroughout epithelial layers as well as binds to the nega-tive valences of mucopolysaccharides [19]; this will de-crease the availability of TB for the staining of the urinarytract. In addition, thiazine dyes like TB and MB can bereduced in vivo by a variety of enzymatic and nonenzy-matic reductants [18, 20–22]. The reduction of theirstrongly polar, relatively hydrophilic blue forms to theircolorless, highly lipophilic forms (leuco-TB and -MB) byintra- and extracellular electron donors increases theirtissue uptake [18, 20]. In the isolated perfused rabbit lung,toluidine blue O was reduced faster than MB by the pul-monary endothelium and thus more extracted from theperfusate by the lung tissue [20]. Reduction rate wasseveralfolds that for steady state sequestration and wasinversely proportional to the flow rate of the perfusate.Tissue uptake may be increased under hyperoxic condi-tions (as in the present study) due to enhanced intracellularreoxidation and thus trapping of the oxidized dyes. Intra-cellularly untrapped, still reduced dyes can return to theperfusate and be reoxidized (redox cycling). When molec-ular oxygen is reduced this way, superoxide (O2·

−) isformed [21]. Presumably, also other endothelial cells canreduce TB because reduction of electron acceptors on cellsurfaces by reductases is a well-known phenomenon[22–24]. In another study, a significant decrease in theclearance rate of total TB and a marked increase in therate of urinary elimination of leuco-TB was observedwhen TB was administered in sheep with methemoglobi-nemia [18], i.e., at methemoglobinemia a weaker stainingof the ureters is expected.

As indicated by the studies above, both the kinetics andthe intensity of the ureter staining probably correlate nega-tive inversely with the degree of colorless leuco-TB

formation, while the rate of reduction probably dependson the rate of administration, the microvascular flow, andthe blood pressure. When TB is infused within 10 min, theintravascular concentration is lower and there is more timefor its reduction/tissue uptake until it reaches the kidneysthan after its 1-min injection. The effect of TB reduction onthe kinetics of ureter staining appears to be especiallyapparent when lower doses are infused, while at higherones the reducing system is increasingly overwhelmed(Fig. 5a). The 1-min injection of higher doses appears torapidly impair kidney function, probably due to the strongerdecreased blood pressure and microvascular flow. Also forthis reason, the injection of ≥1.33 mg TB/kg is not wellsuitable for ureter staining, while the injection of lowerdoses should be more appropriate than their infusion.

Recently, a method based on the near-infrared fluores-cence properties of MB was introduced, allowing to identifyureters intraoperatively (open and laparoscopic surgeries onpigs) in real-time after intravenous MB administration [25].MB was used in that study as it is, unlike in Europe,approved in the USA for parenteral administration and thusclinically available [3, 25]. At open surgery, doses of0.1 mg/kg MB provided prolonged imaging of the uretersand a dose of 0.5 mg/kg provided statistically significantimprovement of contrast-to-background ratio. For the real-time ureteral identification during laparoscopic surgery,however, 1 mg MB/kg was necessary to produce an ac-ceptable staining of the ureters. Unlike in our study withTB, there was little difference in MB performance whenthe dye was given as a rapid bolus or an infusion over5 min [25]. This may be explained by the shorter infusionperiod (5 vs. 10 min) and the faster reduction of TB [20].The main advantage of the method of Matsui et al. com-pared to the present one is its higher sensitivity due to thefluorescence-based approach, allowing to reduce the min-imum required dose for sufficient urethral imaging. Here-by, of course, the risk of possible side effects is reduced.Disadvantages of a fluorescence-based approach are thatsome technical equipment are required for MB excitationand the emission recordings, and that the red excitationlight can imbue the surgical field with a reddish tint whichmay irritate the surgeon [25].

Conclusion

If our results obtained in rats can be generalized to humans,the intravenous administration of 0.4–0.8 mg TB/kg (30–60 mg TB/75 kg) should be sufficient for the intraoperativestaining of the human urinary tract and thus be the safestdose for this issue. Interestingly, Zieger et al. [5] proposedthat the clinical cardiovascular complications after intrave-nous TB administration simply result from the fact that the

992 Langenbecks Arch Surg (2012) 397:983–993

same familiar volume of the previously used MB (5 mg/ml)was also administered when TB (30 mg/ml) was applied.This would mean that a six-fold higher dose of TB wasadministered to stain the urinary tract than intended, i.e.,150–300 mg/patient instead of 25–50 mg, which is close tothe dose we recommend as sufficient and safe here.

Before TB is administered, the QT interval should bechecked and electrolyte levels optimized [3]. TB should notbe administered via a central venous catheter but injectedunder ECG monitoring within a minute through a peripheralvenous access. Due to the numerous variables that influencethe presentation of the urinary tract with MB and TB, and inlight of their side effects, thiazine dyes are not very suitable forthis task. The development of appropriate dyes is desirable.

Acknowledgments We would like to thank Mr. Falk Kaehler, whoexcellently performed the experiments and respective tests, and Mr.Manfred Schmidtmann (Institut für Physiologie, Universität Duisburg-Essen) for his technical assistance regarding the ECG measurements.This work was supported by Dr. F. Köhler Chemie (Bensheim,Germany).

Conflicts of interest None.

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