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Shivering, which usually occurs as a thermoregulatory response to cold, may also occur following general or neuraxial anesthesia. Some of the causative factors of this type of shivering may be common to both, but some are particular to neuraxial anesthesia. Although shivering may have beneficial thermoregulatory effects, it places the body under increased physiological stress. In a broad sample of 21 studies, the median incidence of shivering related to neuraxial anesthesia in the control groups was 55%. Both pharmacological and nonpharmacological mechanisms have been found to be effective in reducing this shivering. This review aims to elucidate the mechanisms of the shivering that occurs during neuraxial anesthesia, and to examine strategies for prevention and treatment of this shivering.

Cytochrome b5 reductase (cb5r), a member of the flavoprotein transhydrogenase family of oxidoreductase enzymes, catalyzes the transfer of reducing equivalents from the physiological electron donor, NADH, to two molecules of cytochrome b5. We have determined the correct nucleotide sequence for the putative full-length, membrane-associated enzyme from Canis familiaris, and have generated a heterologous expression system for production of a histidine-tagged variant of the soluble, catalytic diaphorase domain, comprising residues I33 to F300. Using a simple two-step chromatographic procedure, the recombinant diaphorase domain has been purified to homogeneity and demonstrated to be a simple flavoprotein with a molecular mass of 31,364 (m/z) that retained both NADH:ferricyanide reductase and NADH:cytochrome b5 reductase activities. The recombinant protein contained a full complement of FAD and exhibited absorption and CD spectra comparable to those of a recombinant form of the rat cytochrome b5 reductase diaphorase domain generated using an identical expression system, suggesting similar protein folding. Oxidation-reduction potentiometric titrations yielded a standard midpoint potential (Eo') for the FAD/FADH2 couple of -273+/-5 mV which was identical to the value obtained for the corresponding rat domain. Thermal denaturation studies revealed that the canine domain exhibited stability comparable to that of the rat protein, confirming similar protein conformations. Initial-rate kinetic studies revealed the canine diaphorase domain retained a marked preference for NADH versus NADPH as reducing substrate and exhibited kcat's of 767 and 600 s(-1) for NADH:ferricyanide reductase and NADH:cytochrome b5 reductase activities, respectively, with Km's of 7, 8, and 12 microM for NADH, K3Fe(CN)6, and cytochrome b5, respectively. Spectral-binding constants (Ks) determined for a variety of NAD+ analogs indicated the highest and lowest affinities were observed for APAD+ (Ks=71 microM) and PCA+ (Ks=>31 mM), respectively, and indicated the binding contributions of the various portions of the pyridine nucleotide. These results provide the first correct sequence for the full-length, membrane-associated form of C. familiaris cb5r and provide a direct comparison of the enzymes from two phylogenetic sources using identical expression systems that indicate that both enzymes have comparable spectroscopic, kinetic, thermodynamic, and structural properties.

The clinical disorder of recessive congenital methemoglobinemia (RCM, OMIN 250800) is associated with mutations in NADH:cytochrome b5 reductase (cb5r) and manifests as cyanosis from birth. Screening a cyanotic infant indicated elevated methemoglobin levels and decreased cb5r activity suggesting RCM. Sequencing the DIA1 gene encoding cb5r revealed a novel mutation, C27161T (NCBI accession number: NT_011520), resulting in replacement of proline at amino acid 275 with leucine (P275L). To understand how this mutation would affect cb5r's function, the P275L variant was expressed in a heterologous expression system and spectroscopic, thermodynamic, and thermostability studies were performed. The leucine substitution at residue 275 was found to significantly decrease the affinity towards the physiological reducing substrate, NADH, without affecting the activity of the P275L variant. From the rat model, residue 275 is predicted to be part of a conserved "CGPPPM" motif important for the binding and correct positioning of the NADH reducing substrate. Thus P275 influences the interaction with NADH which was confirmed by the change in affinity towards the physiological reducing substrate.

NADH-cytochrome b(5) reductase deficiency results clinically in either type I or type II recessive congenital methemoglobinemia. The more severe type II form is associated with a global deficiency of cytochrome b(5) reductase and is characterized by cyanosis with neurological dysfunction. In contrast, the only symptom for type I is cyanosis. We have identified a novel G to A mutation at position 15,635 in the DIAI gene of a 4-month-old baby that results in a glycine to serine substitution at codon 75 in the cytochrome b(5) reductase protein. The G75S mutation, located in the FAD-binding lobe of cytochrome b(5) reductase, was found in association with the previously described V252M variant. The V252M mutation is present in the NADH-binding domain and associated with both types I and II recessive congenital methemoglobinemia. Since the G75S and V252M mutations represent radical changes in differing regions of cytochrome b(5) reductase, generating and characterizing these variants singly and in combination using a rat heterologous expression system would provide insight into the differences between types I and II disease at the molecular level. Although all three variants were found to retain stoichiometric levels of FAD with spectroscopic and thermodynamic properties comparable to those of native cytochrome b(5) reductase, all exhibited decreased catalytic efficiency and reduced protein stability reflecting the position of the mutations in the primary structure. The G75S variant retained only 11% of the catalytic efficiency of the wild-type enzyme. Thus, cytochrome b(5) reductase deficient patients who are heterozygous for either FAD- or NADH-binding lobe mutations can exhibit the clinically less severe type I phenotype.

Cytochrome b5 reductase (cb5r), a member of the ferredoxin:NADP+ reductase family of flavoprotein transhydrogenases, catalyzes the NADH-dependent reduction of cytochrome b5. Within this family, a conserved "GxGxxP" sequence motif has been implicated in binding reduced pyridine nucleotides. However, Glycine 179, a conserved residue in cb5r primary structures, precedes this six-residue "180GxGxxP185" motif that has been identified as binding the adenosine moiety of NADH. To investigate the role of G179 in NADH complex formation and NAD(P)H specificity, a series of rat cb5r variants were generated, corresponding to G179A, G179P, G179T, and G179V, recombinantly expressed in Escherichia coli and purified to homogeneity. Each mutant protein was found to incorporate FAD in a 1:1 cofactor/protein stoichiometry and exhibited absorption and CD spectra that were identical to those of wild-type cb5r, indicating both correct protein folding and similar flavin environments, while oxidation-reduction potentials for the FAD/FADH2 couple (n = 2) were also comparable to the wild-type protein (E(o)' = -272 mV). All four mutants showed decreased NADH:ferricyanide reductase activities, with kcat decreasing in the order WT > G179A > G179P > G179T > G179V, with the G179V variant retaining only 1.5% of the wild-type activity. The affinity for NADH also decreased in the order WT > G179A > G179P > G179T > G179V, with the Km(NADH) for G179V 180-fold greater than that of the wild type. Both Ks(H4NAD) and Ks(NAD+) values confirmed that the G179 mutants had both compromised NADH- and NAD+-binding affinities. Determination of the NADH/NADPH specificity constant for the various mutants indicated that G179 also participated in pyridine nucleotide selectivity, with the G179V variant preferring NADPH approximately 8000 times more than wild-type cb5r. These results demonstrated that, while G179 was not critical for either flavin incorporation or maintenance of the appropriate flavin environment in cb5r, G179 was required for both effective NADH/NADPH selectivity and to maintain the correct orientation and position of the conserved cysteine in the proline-rich "CGpppM" motif that is critical for optimum NADH binding and efficient hydride transfer.

Type I recessive congenital methaemoglobinaemia (RCM), caused by the reduced form of nicotinamide adenine dinucleotide (NADH)-cytochrome b(5) reductase (cytb(5)r) deficiency, manifests clinically as cyanosis without neurological dysfunction. Two mutations, E255- and G291D, have been identified in the NADH-binding lobe of cytb(5)r in previously reported patients, and we have detected a further novel mutation, D239G, in this lobe in two unrelated Irish families. Although one family belongs to the genetically isolated Traveller Community, which separated from the general Irish population during the 1845-48 famine, the D239G mutation was present on the same haplotype in both families. Three known cytb(5)r mutations were also identified, including the R159- mutation, which causes loss of the entire NADH-binding lobe and had previously been reported in an individual with type II RCM. Characterization of the three NADH-binding lobe mutants using a heterologous expression system revealed that all three variants retained stoichiometric levels of flavin adenine dinucleotide with spectroscopic and thermodynamic properties comparable with those of native cytb(5)r. In contrast to the E255- and G291D variants, the novel D239G mutation had no adverse impact on protein thermostability. The D239G mutation perturbed substrate binding, causing both decreased specificity for NADH and increased specificity for NADPH. Thus cytb(5)r deficient patients who are heterozygous for an NADH-binding lobe mutation can exhibit the clinically less severe type I phenotype, even in association with heterozygous deletion of the NADH-binding lobe.

The conserved sequence motif "RxY(T)(S)xx(S)(N)" coordinates flavin binding in NADH:cytochrome b(5) reductase (cb(5)r) and other members of the flavin transhydrogenase superfamily of oxidoreductases. To investigate the roles of Y93, the third and only aromatic residue of the "RxY(T)(S)xx(S)(N)" motif, that stacks against the si-face of the flavin isoalloxazine ring, and P92, the second residue in the motif that is also in close proximity to the FAD moiety, a series of rat cb(5)r variants were produced with substitutions at either P92 or Y93, respectively. The proline mutants P92A, G, and S together with the tyrosine mutants Y93A, D, F, H, S, and W were recombinantly expressed in E. coli and purified to homogeneity. Each mutant protein was found to bind FAD in a 1:1 cofactor:protein stoichiometry while UV CD spectra suggested similar secondary structure organization among all nine variants. The tyrosine variants Y93A, D, F, H, and S exhibited varying degrees of blue-shift in the flavin visible absorption maxima while visible CD spectra of the Y93A, D, H, S, and W mutants exhibited similar blue-shifted maxima together with changes in absorption intensity. Intrinsic flavin fluorescence was quenched in the wild type, P92S and A, and Y93H and W mutants while Y93A, D, F, and S mutants exhibited increased fluorescence when compared to free FAD. The tyrosine variants Y93A, D, F, and S also exhibited greater thermolability of FAD binding. The specificity constant (k(cat)/K(m)(NADH)) for NADH:FR activity decreased in the order wild type > P92S > P92A > P92G > Y93F > Y93S > Y93A > Y93D > Y93H > Y93W with the Y93W variant retaining only 0.5% of wild-type efficiency. Both K(s)(H4NAD) and K(s)(NAD+) values suggested that Y93A, F, and W mutants had compromised NADH and NAD(+) binding. Thermodynamic measurements of the midpoint potential (E degrees ', n = 2) of the FAD/FADH(2) redox couple revealed that the potentials of the Y93A and S variants were approximately 30 mV more positive than that of wild-type cb(5)r (E degrees ' = -268 mV) while that of Y93H was approximately 30 mV more negative. These results indicate that neither P92 nor Y93 are critical for flavin incorporation in cb(5)r and that an aromatic side chain is not essential at position 93, but they demonstrate that Y93 forms contacts with the FAD that effectively modulate the spectroscopic, catalytic, and thermodynamic properties of the bound cofactor.

Recessive congenital methemoglobinemia (RCM, OMIM 250800) arises from defects in either the erythrocytic or microsomal forms of the flavoprotein, cytochrome b5 reductase (cb5r) and was the first disease to be directly associated with a specific enzyme deficiency. Of the 33 verified mutations in cb5r that give rise to either the type I (erythrocytic) or type II (generalized) forms of RCM, three of the mutations, corresponding to P144L, L148P, and R159*, are located in a segment of the primary sequence composed of residues G143 to V171 which serves as a "hinge" or "linker" region between the FAD- and NADH-binding lobes of the protein. With the exception of R159*, which produces a truncated non-functional cb5r resulting in type II RCM, the type I methemoglobinemias resulting from the P144L or L148P mutations have been proposed to be due to decreased enzyme stability. Utilizing a recombinant form of the rat cb5r enzyme, we have generated the P144L, L148P, and P144L/L148P mutants, purified the resulting proteins to homogeneity and characterized their spectroscopic, kinetic, and thermodynamic properties. The three mutant proteins retained full complements of FAD with the P144L and L148P variants being spectroscopically indistinguishable from wild-type cb5r. In contrast, kinetic analyses revealed that the P144L, L148P, and P144L/L148P variants retained only 28, 31, and 8% of wild-type NADH:cytochrome b5 reductase activity, respectively, together with significant alterations in affinity for both NADH and NAD+. In addition, FAD oxidation-reduction potentials were 32, 19, and 65 mV more positive for the mutants than the corresponding FAD/FADH2 couple in native cb5r (E0'=-272 mV). Thermal and proteolytic stability measurements indicated that all three mutants were less stable than the wild-type protein while differential spectroscopy indicated altered pyridine nucleotide binding in all three variants. These results demonstrate that the "hinge" region is important in maintaining the correct orientation of the flavin- and pyridine nucleotide-binding lobes within the protein for efficient electron transfer and that the P144L and L148P mutations disrupt the normal registration of the FAD- and NADH-binding lobes resulting in altered affinities for both the physiological reducing substrate, NADH and its product, NAD+.