Charles B. Grissom

Minimally invasive lymphatic mapping using fluorescently labeled vitamin B12

BACKGROUND We examined the usefulness of a new agent in the mapping and dissection of inguinal lymph nodes in the pig. Cy5-cobalamin bioconjugate is blue under visible light and fluoresces brilliant red with laser stimulation. The wavelength of the emitted red light is sufficiently long that it is visible through blood, subcutaneous fat, and fascia. Currently available surgical techniques of minimally invasive dissection are well suited for using fluorescent detection in a dark operating field with minimal modification of an existing Hopkins surgical telescope. MATERIALS AND METHODS We tested this concept in the live post-adolescent, female, nonlactating pig (30 kg). We insufflated the subcutaneous tissue over the groin and inserted three ports (1 x 10 mm and 2 x 5 mm) for dissection. We injected the Cy5-cobalamin bioconjugate in a dermal location on the hind limb. A HeNe laser stimulated the CobalaFluor in the lymphatics and the emitted fluorescence passed through a holographic notch filter to a three-chip camera. RESULTS Under white light, the lymphatic trunks and the sentinel node were visualized within minutes of injection. Both the lymphatic trunks and the node fluoresced bright red under stimulation with red laser light. CONCLUSIONS These preliminary studies establish the potential usefulness of this new agent in lymphatic mapping. This novel technology might be useful in visualizing cancers that spread to regional lymph nodes. This technique has the potential to map the lymphatic drainage and to identify the presence of malignant cells in that drainage with currently available minimally invasive technology.

Application of a fluorescent cobalamin analogue for analysis of the binding kinetics: A study employing recombinant human transcobalamin and intrinsic factor

Fluorescent probe rhodamine was appended to 5′ OH‐ribose of cobalamin (Cbl). The prepared conjugate, CBC, bound to the transporting proteins, intrinsic factor (IF) and transcobalamin (TC), responsible for the uptake of Cbl in an organism. Pronounced increase in fluorescence upon CBC attachment facilitated detailed kinetic analysis of Cbl binding. We found that TC had the same affinity for CBC and Cbl (Kd = 5 × 10−15 m), whereas interaction of CBC with the highly specific protein IF was more complex. For instance, CBC behaved normally in the partial reactions CBC + IF30 and CBC + IF20 when binding to the isolated IF fragments (domains). The ligand could also assemble them into a stable complex IF30–CBC–IF20 with higher fluorescent signal. However, dissociation of IF30–CBC–IF20 and IF–CBC was accelerated by factors of 3 and 20, respectively, when compared to the corresponding Cbl complexes. We suggest that the correct domain–domain interactions are the most important factor during recognition and fixation of the ligands by IF. Dissociation of IF–CBC was biphasic, and existence of multiple protein–analogue complexes with normal and partially corrupted structure may explain this behaviour. The most stable component had Kd = 1.5 × 10−13 m, which guarantees the binding of CBC to IF under physiological conditions. The specific intestinal receptor cubilin bound both IF–CBC and IF–Cbl with equal affinity. In conclusion, the fluorescent analogue CBC can be used as a reporting agent in the kinetic studies, moreover, it seems to be applicable for imaging purposes in vivo.

The origin of magnetic field dependent recombination in alkylcobalamin radical pairs.

Magnetic field effect studies of alkylcobalamin photolysis provide evidence for the formation of a reactive radical pair that is born in the singlet spin state. The radical pair recombination process that is responsible for the magnetic field dependence of the continuous‐wave (CW) quantum yield is limited to the diffusive radical pair. Although the geminate radical pair of adenosylcob(III)alamin also undergoes magnetic field dependent recombination (A. M. Chagovetz and C. B. Grissom, J. Am. Chem. Soc. 115, 12152–12157, 1993), this process does not account for the magnetic field dependence of the CW quantum yield that is only observed in viscous solvents. Glycerol and ethylene glycol increase the microviscosity of the solution and thereby increase the lifetime of the spin‐correlated diffusive radical pair. This enables magnetic field dependent recombination among spin‐correlated diffusive radical pairs in the solvent cage. Magnetic field dependent recombination is not observed in the presence of nonviscosigenic alcohols such as isopropanol, thereby indicating the importance of the increased microviscosity of the medium. Paramagnetic radical scavengers that trap alkyl radicals that escape the solvent cage do not diminish the magnetic field effect on the CW quantum yield, thereby ruling out radical pair recombination among randomly diffusing radical pairs, as well as excluding the involvement of solvent‐derived radicals. Magnetic field dependent recombination among alkylcobalamin radical pairs has been simulated by a semiclassical model of radical pair dynamics and recombination. These calculations support the existence of a singlet radical pair precursor.

Magnetic Field Effects in Model B12 Enzymatic Reactions. the Photolysis of Methylcob(III)Alamin

— 2000 Gauss vermindert. Dies ist konsistent damit, daB das geminale Radikalpaar im elektronischen Singulettzustand gebildet wird. In waBrigen Lösungen, die ein schnelles Auseinanderdiffundieren (Escape) des spinkorrelierten Radikalpaares erlauben, wird kein signifikanter Magnetfeldeffekt beobachtet. Glyzerin kann zur Erhöhung der Mikroviskositàt und Verlangsamung der molekularen Diffusion verwendet werden. Ficoll-400, ein makromolekulares Polymer

Use of magnetic field effects to study coenzyme B12-dependent reactions

Publisher Summary This chapter summarizes the theory of magnetic field effects (MFEs) on chemical and enzymatic reactions and outlines the experimental methods necessary to use this powerful technique to study a variety of enzymatic and chemical reactions with radical pair (RP) intermediates. There are at least three mechanisms by which a magnetic field can change the rate of intersystem crossing in radical pair intermediates—the hyperfine interaction mechanism, the Δg mechanism, and the level crossing mechanism. The use of magnetic field effects to probe enzymatic reactions is usually limited to static (DC) magnetic fields that do not vary with time. Magnetic field effects provide a new method to study biological reactions with radical pair intermediates. Coenzyme B 12 -dependent enzymes with radical pair intermediates are well suited for study by this technique, as the RP is reversibly formed and the hyperfine interactions from the paramagnetic Co(II) provide a mechanism by which magnetic field-dependent intersystem crossing can occur. Not all enzymes with radical intermediates exhibit magnetic field-dependent chemistry—only the subset of enzymes with a pair of radicals.

Uptake and distribution of fluorescently labeled cobalamin in neoplastic and healthy breast tissue

Fluorescent analogs of cobalamin (vitamin B12) have been developed as diagnostic markers of cancer cells. These compounds are recognized by transcobalamin, a cobalamin transport protein, with high affinity, as shown by surface plasmon resonance. The cellular sequestration and gross distribution of fluorescent cobalamin bioconjugates in breast tissue is being examined by epifluorescence microscopy. The distribution of each compound is being evaluated in proliferative and non-proliferative tissue, i.e. normal tissue and breast carcinoma. The results of preliminary studies suggest that fluorescent analogs of cobalamin may be a useful tool in therapeutic breast operations to define tumor margins and to distinguish neoplastic breast tissue from healthy breast tissue.