Preliminary investigation of near-infrared spectroscopic measurements of urea, creatinine, glucose, protein, and ketone in urine.

OBJECTIVE: We investigated the use of near-infrared spectroscopy as an analytical tool to quantify concentrations of urea, creatinine, glucose, ketone, and protein in urine. DESIGN AND METHODS: FT-IR spectroscopy in conjunction with a polynomial based spectral smoothing method was applied to urine specimens. A partial factorial experimental design was employed to collect spectra using normal and spiked urine samples. RESULTS: Our results show that the spectral signatures of urea, creatinine, glucose, ketone, and protein in the 1350 to 1800 nm and 2050 to 2375 nm range are sufficiently strong and unique for accurate measurements. CONCLUSIONS: The accuracy of near infrared for quantifying concentrations of urea and creatinine is only slightly less than our selected reference methods. Glucose, ketone and protein are sufficiently accurate to be useful as a screening tool for wellness. The method successfully accounts for biologic matrix variation. The advantages of near-infrared analysis are (1) no reagents, (2) ease of sample preparation, (3) speed, and (4) the ability to quantify multiple analytes with one spectra.

Noninvasive determination of hemoglobin and hematocrit using a temperature-controlled localized reflectance tissue photometer.

We performed visible/near-infrared optical measurements on the forearm of human subjects. We conducted four studies: one study using a commercial diffuse reflectance spectrometer, and three studies using a breadboard temperature-controlled localized reflectance tissue photometer. Calibration relationships were established between skin reflectance signal and either the reference blood hemoglobin (Hb) concentration or the hematocrit values (Hct). Prediction results were expressed as the prediction correlation coefficient (r(p)) and the standard error for cross-validation prediction (CV-SEP). Using diffuse reflectance measurement, r(p) = 0. 8, CV-SEP = 0.9 g/dL for Hb and r(p) = 0.7, CV-SEP = 3.3% for Hct (n = 40). In a localized reflectance study involving calculating the absorption and scattering coefficients and including effect of change of skin temperature in the calibration model, the best prediction results were r(p) = 0.9, CV-SEP = 0.8 g/dL for Hb and r(p) = 0.8, CV-SEP = 3% for Hct (n = 26). In a second localized reflectance study on a population having diverse skin colors at 34 degrees C, the optimal model led to r(p) = 0.8, CV-SEP = 0.9 g/dL for Hb and r(p) = 0.9, CV-SEP = 2.1% for Hct (n = 28) using the localized reflectance values without deducing the absorption and scattering coefficients. Improvement in the correlation was more noticeable for Hct than for the case of Hb. The photometer was used to screen prospective blood donors with low Hb concentration. It was possible to predict anemic subjects in the limited prospective blood donor population.

Crystallographic analysis of acrosomal bundle from Limulus sperm.

The acrosomal process of Limulus sperm contains a bundle of filaments composed of actin and a 102 kDa protein in a 1:1 molar ratio. The structure of the bundle in true discharge was investigated by electron cryomicroscopy, X-ray scattering and crystallographic image analysis. A bundle can be characterized as a quasi-crystal with continuously varying views along the bundle axis. Each segment of the bundle is found to obey the symmetry of space group P1, with a = b = 147 A, c = 762 A, alpha = 90 degrees, beta = 90.6 degrees, gamma = 120 degrees. A unit cell contains a helical repeat of the filament with a selection rule following that of an actin filament. A 24 A projection map based on the h0l view was reconstructed after averaging 5300 unit cells from six electron images. Filaments in this projection are well separated and clearly display a 21 screw symmetry. This screw symmetry results from the helical parameters of the bundle filament and is found to be a non-crystallographic symmetry element present in the unit cell. Our structural analysis has led to the proposal that the assembly of a stable bundle with a defined maximum diameter can be controlled by the crystallographic packing of the twisted filaments.

Estimation of allowable errors for tilt parameter determination in protein electron crystallography.

A three-dimensional reconstruction of a thin protein crystal requires an accurate assignment of amplitudes and phases in the three-dimensional reciprocal space. This assignment depends upon the tilt parameters which are experimentally determined. A theoretical estimate is made of the tolerable error in the determination of tilt parameters as a function of resolution and crystal thickness.

Visualization of alpha-helices in tobacco mosaic virus by cryo-electron microscopy.

We have used tobacco mosaic virus (TMV) as a test specimen, in order to develop techniques for the analysis of high-resolution structural detail in electron micrographs of biological assemblies with helical symmetry. It has previously been shown that internal details of protein structure can be visualized by processing electron micrographs of unstained specimens of extended two-dimensional crystalline arrays. However, the techniques should in principle be applicable to other periodic specimens, such as assemblies with helical symmetry. We show here that data to spacings better than 10 A can be retrieved from electron images of frozen hydrated TMV. The three-dimensional computed map agrees well with that derived from X-ray diffraction and shows the two pairs of alpha-helices forming the core of the coat subunit, the C alpha-helix and the viral RNA. The results demonstrate that it is possible to determine detailed internal structure in helical particles.

Cryo electron microscopy of unstained, unfixed RecA-cssDNA complexes.

Complexes of RecA protein with phi X174 circular single-stranded DNA (cssDNA) with and without ATP gamma S were rapidly frozen and embedded in a thin layer of vitreous ice. The electron micrographs of these frozen-hydrated complexes clearly show visible helicity. Quantitative image analyses of these micrographs reveal the helical pitch and the axial rise between DNA bases of these complexes. Both of these structural parameters of RecA-cssDNA complexes increase significantly when ATP gamma S is present. These observations agree qualitatively but not quantitatively with those from negative stained specimens and confirm the general model that the interactions among RecA molecules and between RecA and DNA could change according to the functional states of the RecA-cssDNA complex.

Containment system for the preparation of vitrified-hydrated virus specimens.

A guillotine-type quick freezing device and a bio-hazard containment box have been designed, constructed, and used to prepare vitrified-hydrated specimens of viruses in their native environment. Special design considerations include the preservation of the specimen in its natural state in vitrified ice, prevention of virus aerosols escape, and control of the potentially explosive air-coolant vapor mixtures.

High resolution cryo system designed for JEM 100CX electron microscope.

A high resolution (3.5 A) cold stage operated at 123 K and an improved anti-contamination device have been constructed and operated in a top-entry JEM 100CX electron microscope. High resolution electron diffraction patterns and images of hydrated tobacco mosaic virus particles in vitrified ice have been recorded with the use of this cryo system.

Quantitative assessment of radiation damage in a thin protein crystal.

Radiation damage is a limiting factor for high resolution structural determination of protein crystals. We have used the median and quartile values of Ne exposures and the structural disordering factor (delta B) to describe the crystalline disordering due to radiation damage in thin crystals of crotoxin complex embedded in glucose at room and low temperatures and in ice embedded crystals. By approximating the radiation damage to follow first order kinetics, we applied a correction to the diffraction intensities from patterns with high accumulated exposures. We used the structural similarity factor (R) to show that, for some data, the effectiveness of the correction can be as good as those in protein X-ray crystallography.

Electron imaging of crotoxin complex thin crystal at 3.5 A.

Crotoxin complex forms thin crystals which are suitable for electron crystallographic analysis. We have used a 100 kV electron microscope equipped with a superconducting lens to image this crystal embedded in glucose. Optical diffraction analysis of the micrographs show unambiguously a structural resolution of 3.9 A which has not been obtained with the conventional microscope at room temperature. A density map with a nominal resolution of 3.5 A has been synthesized from these images by computer processing techniques.

Specimen preparative methods for electron crystallography of soluble proteins.

Technical factors which influence the choice of specimen preparation method for electron crystallographic study of thin crystals of soluble proteins are discussed. Ice embedding appears to be the most desirable choice of preparation method. However, in terms of the yield of major structural information, we conclude that negative stain remains a useful method for low resolution and glucose embedding for high resolution.

Experimental strategy in three-dimensional structure determination of crotoxin complex thin crystal.

Electron images and diffraction patterns of crotoxin complex thin crystals contain high-resolution structural information in projection at both room and low temperatures. This paper outlines our strategy for collecting three-dimensional amplitude and phase data from these uniquely well-ordered toxin thin crystals. Preliminary experiments in support of the feasibility of this approach are presented.

Low dose electron microscopy of the crotoxin complex thin crystal.

The crotoxin complex from Crotalus d. terrificus rattlesnake venom was crystallized in the form of thin platelets. These crystals were prepared by the glucose embedding technique and examined by low dose electron microscopy. Electron diffraction patterns and images have been recorded to 2.2 and 4.5 A, respectively. By a combination of electron and X-ray diffraction techniques, the space group of this crystal was determined to be P4(2)22 with eight crotoxin complex molecules in one unit cell with dimensions of 38.8 A x 38.8 A x 256.8 A. The Patterson maps and the symmetry reliability factors calculated from the electron diffraction intensities clearly showed the existence of three types of electron diffraction patterns in different crystals. The phases in the computer-calculated transform of the low dose images also show the variation in symmetry among crystals. These phenomena are explained by the presence of crystals consisting of one-half, three-quarter and one unit cell in thickness. The interpretation of the computer reconstructed two-dimensional density map was limited, partly because of the similarity in density between the protein and the embedding glucose and partly because of the non-uniqueness in relating projected structure to the three-dimensional structure.

Postsynaptic effects of crotoxin and of its isolated subunits.

Crotoxin is a potent neurotoxin from the venom of Crotalus durissus terrificus. It is composed of two subunits: a basic phospholipase A2 with low toxicity (component B) and an acidic protein seemingly devoid of intrinsic biological activity (component A). Crotoxin and its isolated phospholipase subunit block the depolarisation caused by cholinergic agonists on the isolated electroplaque from Electrophorus electricus. The other component, which is inactive when applied alone, enhances the pharmacological activity of the phospholipase when the two components are used together. Crotoxin also blocks the increase of 22Na+ efflux caused by carbamylcholine from excitable microsacs prepared from Torpedo marmorata electric organ. Crotoxin therefore acts postsynaptically, but does not interfere with the binding of alpha-toxin from Naja nigricollis to the nicotinic cholinergic receptor site. Instead, like local anesthetics, it stabilizes a desensitized form of the acetylcholine receptor characterized by its high affinity for agonists. The phospholipase component B binds in a non-saturable manner to receptor-rich membranes. In contrast, component A does not bind to acetylcholine receptor-rich membranes, but completely prevents the non-saturable binding of component B. When the two components are applied together, a saturable binding of the latter is observed with the acetylcholine receptor-rich membranes.

Search for relationships among the hemolytic, phospholipolytic, and neurotoxic activities of snake venoms.

Several snake venom neurotoxins are larger and more complex than the well-studied group of postsynaptic toxins exemplified by alpha-bungarotoxin. Several of these, exemplified by beta-bungarotoxin, show phospholipase A2 activity (phosphatide 2-acylhydrolase, EC 3.1.1.4) when tested in the presence of detergents. The high hemolytic activity of crotoxin, the neurotoxin of Crotalus durissus terrificus, in the presence of lecithin has been attributed to this activity. The phospholipase A2 activity of several snake venom proteins has now been compared under the physiological conditions of the hemolysis tests. It appears that only the basic component of crotoxin, B, is enzymatically active, and that its activity is not inhibited by component A under these conditions, or in the presence of deoxycholate. Phosphatidylserine is found to be digested more readily than egg white phosphatidylcholine; and also causes hemolysis in conjunction with much lower levels of crotoxin. In neither case is calcium required or stimulating. Phospholipase from Crotalus adamanteus, which is not neurotoxic, digests phosphatidylcholine more rapidly than does crotoxin, but phosphatidylserine more slowly; yet it is slightly less active than crotoxin in the hemolysis test with phosphatidylcholine, and much less with phosphatidylserine. The digestion of several phospholipids by either enzyme fails to release the expected protons in the absence of detergents at 37 degrees .beta-Bungarotoxin, highly neurotoxic, has negligible phospholipase A2 activity in the absence of detergents, and is almost nonhemolytic in conjunction with all phospholipids tested.Binding studies with (125)I-labeled compounds show that rabbit erythrocytes and ghosts have much greater affinity for crotoxin than for beta-bungarotoxin and do not bind Crotalus adamanteus phospholipase. The crotoxin complex is split in the course of binding, with only component B, the hemolytic component, becoming bound. It appears that the role of component A may be to diminish the nonspecific binding tendency of component B. Our data appear to be consistent with the concepts that affinity to membranes, particularly to specific sites on synaptic membranes, is the critical requirement for beta type neurotoxicity, and that this property, at least in some instances, has evolved from phospholipase A2 enzymes, but does not necessarily require retention and expression of enzymatic activity.