A new biological assay for measuring cyanide in blood.

UNLABELLED: Clinical diagnosis of cyanide poisoning is complicated by the lack of an easy, convenient assay for cyanide concentration in blood. Therapy may be delayed with unconfirmed diagnosis because the conventional antidote to cyanide poisoning exposes patients to substantial risks. We developed a new spectrophotometric assay to measure cyanide by extraction into a sodium hydroxide trap, followed by the addition of exogenous methemoglobin as a colormetric indicator. Samples of blood from 15 healthy subjects and 5 patients who had received prolonged nitroprusside infusions were assayed. To optimize assay characteristics, methemoglobin concentrations, pH, temperature, incubation time, and buffer strengths were varied. Duplicate samples were assayed by using the polarographic method for assay validation. Over a range from 300 ng/mL to 7 microg/mL, the correlation between methods was r = 0.983. Interassay and intraassay variability were 5% and 2%, respectively. Samples drawn from the five patients and tested by using both methods yielded a correlation of r = 0.978. This new assay for cyanide in blood may greatly facilitate the diagnosis and treatment of cyanide ingestion. The use of methemoglobin as the colorimetric indicator in the assay contributes to its low cost and ease of use. IMPLICATIONS: Cyanide, an important factor in death from burn-related inhalation injury, is difficult and time-consuming to measure. We developed a new, rapid blood test for cyanide using methemoglobin as a colormetric indicator. A rapid, accessible test for cyanide may speed the diagnosis and treatment of cyanide poisoning.

On the formation and crystallization of sickle hemoglobin macrofibers.

We have characterized new aspects of macrofiber structure and assembly which provide a mechanism for macrofiber formation from fibers. After the formation of fibers, HbS forms macrofibers by the association of small, organized bundles of partially fused fibers. These macrofibers consist of double strands, packed into antiparallel rows, and are identical to double strands found in crystalline HbS, except that the double strands in macrofibers are axially displaced from their crystalline position and are twisted about the particle axis, whereas in crystals they are linear. In lateral views, electron micrographs of macrofibers show prominent sets of "rows." We use the number of these rows to designate a particular type of macrofiber. In this study we present micrographs of macrofibers with 3 to 11 rows visible in lateral views. Such particles contain from 20 to 200 double strands. The pitch of a macrofiber is coupled to the number of rows in a manner so that the angle between the molecules in the outermost double strand is always 1.8 degrees. This observation has led us to propose that the factor limiting the extent of lateral growth of macrofibers is distortions in bonding between the hemoglobin molecules in the outermost double strands. Similar considerations have provided an explanation of the factors that limit the lateral growth of fibers. Finally, we propose a simple mechanism for the formation of macrofibers from fibers. This mechanism postulates that integral numbers of fibers form specific types of macrofibers and has the virtue of conserving the polarity of the fibers.

On the assembly of sickle hemoglobin fascicles.

Deoxyhemoglobin S fibers associate into bundles, or fascicles, that subsequently crystallize by a process of alignment and fusion. We have used electron microscopy to study the formation of fascicles and the changes in fiber packing that occur during the conversion of fascicles to crystals. The first event in crystallization involves fibers forming fascicles that are initially small and poorly ordered but, with time, become progressively larger and more highly ordered. After six to eight hours, the fibers in a fascicle form a crystalline lattice. The three-dimensional unit cell parameters of this lattice are a = 1300 A, b = 365 A, and c = 210 A (the a axis is parallel to the fiber axis). Fibers have an elliptical cross-section whose major and minor axes are 250 A and 185 A, respectively. When projected on to the unit cell vectors, these dimensions are 210 A and 155 A, so the unit cell dimension of 365 A implies that there are two fibers per unit cell. Theoretically, fibers could pair so that each member of the unit cell is oriented in the same direction (parallel) or opposite directions (antiparallel). Fourier transforms of electron micrographs (or models) cannot distinguish between these alternatives, since the two arrangements produce very similar intensity distributions. The orientation of the fibers was determined from cross-sections of the fascicles in which the fibers are seen end-on. In this view the images of the fibers are rotationally blurred because the fibers twist 30 degrees to 40 degrees about their helical axis through the 300 A to 400 A thick section. We have been able to remove the rotational blur from each of the fibers in the unit cell using the procedures described by Carragher et al. The deblurred images of the two fibers in the unit cell are related by mirror symmetry. This relationship means that the fibers are antiparallel. These observations suggest that crystallization of fibers in fascicles is mediated by assembly of the fibers into antiparallel pairs that contain equal numbers of double strands running in each direction.

Structural analysis of polymers of sickle cell hemoglobin. III. Fibers within fascicles.

We have examined the structure of hemoglobin S fibers, which are associated into large bundles, or fascicles. Electron micrographs of embedded and cross-sectioned fascicles provide an end-on view of the component fibers. The cross-sectional images are rotationally blurred as a result of the twist of the fiber within the finite thickness of the section. We have applied restoration techniques to recover a deblurred image of the fiber. The first step in this procedure involved correlation averaging images of cross-sections of individual fibers in order to improve the signal-to-noise ratio. The rotationally blurred image was then geometrically transformed to polar co-ordinates. In this space, the rotational blur is transformed into a linear blur. The linearly blurred image is the convolution of the unblurred image and a point spread function that can be closely approximated by a square pulse. Deconvolution in Fourier space, followed by remapping to Cartesian co-ordinates, produced a deblurred image of the original micrograph. The deblurred images indicate that the fiber is comprised of 14 strands of hemoglobin S. This result provides confirmation of the fiber structure determined using helical reconstruction techniques and indicates that the association of fibers into ordered arrays does not alter their molecular structure.