Evans blue and gentian violet: Alternatives to methylene blue as a surgical marker dye

Abstract

Dyes, such as methylene blue, are used during vascular surgery to aid in the visualization and orientation of the blood vessel. Although methylene blue dye is believed to be innocuous, we1Barber DA Rubin JW Zumbro GL Tackett RL The use of methylene blue as an extravascular surgical marker impairs vascular responses of human saphenous veins.J THORAC CARDIOVASC SURG. 1995; 109: 21-29Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar recently demonstrated that extravascular application of 1% methylene blue impairs both endothelium-dependent and endothelium-independent vasorelaxation. The present study investigated Evans blue and gentian violet as alternative surgical marker dyes to methylene blue. Human saphenous veins were cut into 2 to 4 mm rings, carefully swabbed with 0.5% Evans blue, 1.0% gentian violet, or Krebs buffer, and incubated in 20 ml of buffer at 25º C for 45 minutes. The vascular rings were then suspended in 10 ml tissue baths containing Krebs buffer, and dose-response curves to acetylcholine, isoproterenol, ve rapamil, and sodium nitroprusside were performed as previously described.1Barber DA Rubin JW Zumbro GL Tackett RL The use of methylene blue as an extravascular surgical marker impairs vascular responses of human saphenous veins.J THORAC CARDIOVASC SURG. 1995; 109: 21-29Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar Vascular rings were assayed in duplicate and averaged to yield a single n value. All statistical analyses were performed with the n values representing the number of patients. Differences were assayed by a one-way analysis of variance followed by a Bonferroni t test for multiple comparisons. All data are presented as the mean ± standard error of the mean. Only differences with a p value < 0.05 were deemed as statistically significant. Potassium chloride (70 mmol/L) produced a contraction of 1.45 ± 0.36 gm in control rings, which was not significantly different from that observed in rings subjected to Evans blue (1.92 ± 0.48 gm) or gentian violet (1.81 ± 0.40 gm). Phenylephrine (10-5 mol/L) also produced a comparable constriction (1.73 ± 0.52 gm), which was unaffected by exposure to either Evans blue or gentian violet–treated tissue. Acetylcholine, which assessed endothelium-dependent responses, produced a dose-dependent vasodilation with the nadir at -54% ± 11%, which was unaffected by exposure to Evans blue (Fig. 1, A). Fig. 1The effect of Evans blue (EB) exposure on vasodilator activity in saphenous veins. Control ring preparations were swabbed with Krebs buffer. Responses are expressed as a percentage of phenylephrine (10-5 mol/L).View Large Image Figure ViewerDownload (PPT)Fig. 1The effect of Evans blue (EB) exposure on vasodilator activity in saphenous veins. Control ring preparations were swabbed with Krebs buffer. Responses are expressed as a percentage of phenylephrine (10-5 mol/L).View Large Image Figure ViewerDownload (PPT)Fig. 1The effect of Evans blue (EB) exposure on vasodilator activity in saphenous veins. Control ring preparations were swabbed with Krebs buffer. Responses are expressed as a percentage of phenylephrine (10-5 mol/L).View Large Image Figure ViewerDownload (PPT)Sodium nitroprusside was used to assess endothelium-independent vasodilation. As seen in Fig. 1, B, 100% relaxation was achieved in response to sodium nitroprusside in control vein segments and segments exposed to Evans blue. Verapamil was used to assess calcium-mediated responses. As seen in Fig. 1, C, no significant differences were noted between control vessels and those treated with Evans blue. Fig. 1, D, shows that the vasodilatory responses to isoproterenol mediated through β2-adrenergic receptors was unaffected by Evans blue treatment. Fig. 1The effect of Evans blue (EB) exposure on vasodilator activity in saphenous veins. Control ring preparations were swabbed with Krebs buffer. Responses are expressed as a percentage of phenylephrine (10-5 mol/L).View Large Image Figure ViewerDownload (PPT)Fig. 1The effect of Evans blue (EB) exposure on vasodilator activity in saphenous veins. Control ring preparations were swabbed with Krebs buffer. Responses are expressed as a percentage of phenylephrine (10-5 mol/L).View Large Image Figure ViewerDownload (PPT) Gentian violet treatment did not affect the vasodilatory responses to acetylcholine (Fig. 2, A), verapamil (Fig. 2, C), or isoproterenol (Fig. 2, D). Fig. 2The effect of gentian violet (GV) exposure on vasodilator activity in saphenous veins. Control ring preparations were swabbed with Krebs buffer. Responses are expressed as a percentage of phenylephrine (10-5 mol/L). *p < 0.05.View Large Image Figure ViewerDownload (PPT)Fig. 2The effect of gentian violet (GV) exposure on vasodilator activity in saphenous veins. Control ring preparations were swabbed with Krebs buffer. Responses are expressed as a percentage of phenylephrine (10-5 mol/L). *p < 0.05.View Large Image Figure ViewerDownload (PPT)Fig. 2The effect of gentian violet (GV) exposure on vasodilator activity in saphenous veins. Control ring preparations were swabbed with Krebs buffer. Responses are expressed as a percentage of phenylephrine (10-5 mol/L). *p < 0.05.View Large Image Figure ViewerDownload (PPT)However, gentian violet inhibited the vasodilatory response to sodium nitroprusside, as seen in panel B of Fig. 2. Fig. 2The effect of gentian violet (GV) exposure on vasodilator activity in saphenous veins. Control ring preparations were swabbed with Krebs buffer. Responses are expressed as a percentage of phenylephrine (10-5 mol/L). *p < 0.05.View Large Image Figure ViewerDownload (PPT)Fig. 2The effect of gentian violet (GV) exposure on vasodilator activity in saphenous veins. Control ring preparations were swabbed with Krebs buffer. Responses are expressed as a percentage of phenylephrine (10-5 mol/L). *p < 0.05.View Large Image Figure ViewerDownload (PPT) The present study demonstrates that Evans blue does not impair either endothelium-dependent or endothelium-independent vasodilation. Alternatively, gentian violet impaired only the endothelial response to sodium nitroprusside, and this degree of impairment was greater than that previously reported with the use of methylene blue dye.1Barber DA Rubin JW Zumbro GL Tackett RL The use of methylene blue as an extravascular surgical marker impairs vascular responses of human saphenous veins.J THORAC CARDIOVASC SURG. 1995; 109: 21-29Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar Methylene blue dye has several actions that could adversely affect vascular reactivity, including inhibition of soluble guanylate cyclase2Ignarro LJ Burke TM Wood KS Wolin MS Kadowitz PJ Association between cyclic GMP accumulation and acetylcholine-elicited relaxation in bovine intrapulmonary artery.J Pharmacol Exp Ther. 1984; 228: 682-690PubMed Google Scholar and production of superoxide.3Marshall JJ Wei EP Lontos HA Independent blockade of cerebral vasodilation from acetylcholine and nitric oxide.Am J Physiol. 1988; 255: H847-H855PubMed Google Scholar The pharmacologic actions of Evans blue and gentian violet have not been studied extensively and thus remain relatively poorly understood. Gentian violet is best known as a topical antimicrobial agent that irreversibly binds microbial deoxyribonucleic acid and directly inhibits replication.4Wakelin LPG Adams A Hunter C Waring MJ Interactions of crystal violet with nucleic acids.Biochemistry. 1981; 20: 5779-5787Crossref PubMed Scopus (41) Google Scholar Evans blue is a biologic stain whose pharmacologic effects have not been extensively studied, but it is commonly used as a diagnostic agent for the determination of plasma volume.510th ed. Evans blue. In The Merck index. Merck & Co, Rahway (NJ)1983: 3855Google Scholar The reason for inhibition of the sodium nitroprusside response by gentian violet is unclear. Gentian violet is metabolized to a reactive intermediate or free radical that could act at the vascular smooth muscle to inhibit the response. However, this is unlikely inasmuch as this mechanism would also interfere with the acetylcholine-mediated vasodilation. A more plausible explanation would be the formation of a reactive intermediate that interacts directly with the sodium nitroprusside molecule. In conclusion, although methylene blue has been used as a marker dye, our studies suggest that it is inappropriate for marking vascular grafts. Evans blue did not adversely affect vasodilation to any agent and thus could be used as an alternative to methylene blue without fear of affecting graft reactivity. Gentian violet also appears to be a better alternate to methylene blue. However, we did observe a decrease in endothelium-independent vasodilation, a factor that should be considered when this agent is used.