Quantitative microanalysis of oligosaccharides by high-performance liquid chromatography

The rapid separation and quantitative determination of per-O-benzoyl oligosaccharides were obtained using high performance, liquid chromatography. Oligosaccharides were completely O-benzoylated without concomitant N-benzoylation of acetamidodeoxyhexoses. Benzoylation prior to analysis allowed a quantitative determination of picomolar amounts because the absorbance at 230 nm of these derivatives is directly proportional to the number of benzoyl groups present. Separation by normal and reversed-phase chromatography was demonstrated, and the best resolution was obtained on an Ultrasphere octyl column. Excellent separations of oligosaccharides containing up to 10 sugar residues present in mannosidosis urine and of malto-oligosaccharides containing up to 15 sugar residues present in Karo syrup were achieved within an analysis time of 30 min. Anomers of maltose, maltotriose, and maltotetraose were separated; for this reason, reduction of complex samples prior to analysis is advisable. The effect of linkage configuration on retention time was tested with reduced, α-linked di- and tri-glucopyranosides. The presence of an acetamidodeoxyhexose residue in an oligosaccharide significantly reduced its retention time, whereas branching had the opposite effect. A linear response was obtained for the injection of 1–600 pmol of raffinose, and the detection limit was 0.5 pmol. Derivatization and analysis of raffinose was shown to yield reproducible results within the range 0.01–1 μmol, and, with special precautions to minimize losses, as little as 100 pmol could be analyzed successfully.     

Quantitative electron probe X-ray microanalysis of thin objects using an independent mass determination method

Elemental mass fraction determinations on thin biological specimens by quantitative electron probe X-ray microanalysis commonly use a total mass estimation based on registration of X-ray continuum. We have tested whether the accurary of such determinations on individual biological objects can be improved when a densitometric method for mass determination is used. Air-dried femtoliter droplets with dry masses in the order of 10⁻¹⁶ kg were used as model objects. An improvement of over two-fold upon the continuum method could be achieved.     

Quantitative electron probe x-ray microanalysis and densitometric mass determination of individual rat blood platelets

An improved method for quantitative electron probe X-ray microanalysis of thin biological specimens, introduced recently, has been applied to the elemental analysis of rat blood platelets. The method uses the X-ray signal to quantify the elemental content of an object and electron scattering to determine the total dry mass of the object. Along with the dry mass distribution, data were obtained on the content and mass fraction of calcium, magnesium, and sulfur in 31 individual platelets. The mean platelet dry mass was found to be 985 fg. The mean Ca, Mg, and S contents were 1.80, 3.41, and 18.3 fg, with mean dry mass fractions of 0.195, 0.396, and 1.96%, respectively. Furthermore, these elements appear to be unevenly distributed among the platelet population.     

Quantitative electron probe microanalysis of leech photoreceptors

Summary The photoreceptive microvilli in the visual cells of the leech protrude into a large intracellular vacuole which is but an extracellular compartment (ionic composition unknown), because it communicates with the extracellular space by narrow ( 20 nm) clefts (septate junctions) of unknown permeability properties. Application of Thiéry's cytochemical silver proteinate method reveals that the vacuole contains carbohydrate-rich material. We used electron probe microanalysis of dry, ultrathin cryosections to determine quantitatively the elemental (K, Na, Cl, Mg, Ca, P, S) composition of the cytoplasm, vacuole and extracellular space.The composition of the vacuole is similar to that of the extracellular space, as shown by the comparable Na/K (11 to 13) and K/Ca (1.8 to 2.2) ratios in these two compartments. There are neglible concentration gradients for Na, K and Cl between vacuole and extracellular space. The vacuole has a high S content and a relatively large deficit of Cl compared to [Na]+[K]+2 [Ca]. Thus the data indicate that the vacuole is in ionic communication with the extracellular space and contains sulfonated glycoprotein(s) that can partially exclude Cl; electroneutrality is maintained in part by these organic anions. The cytoplasmic K concentration (393±30 mmol/kg dry wt) is comparable to that in other nerve cells. The cytoplasmic Cl concentration (216±14 mmol/kg dry wt) is relatively high: significantly (P<0.001) higher than the cytoplasmic Na (130±15 mmol/kg dry wt). The high cytoplasmic Cl content is in excess of that predicted by passive distribution, and suggests the operation of a Cl pump.     

The role of potassium in the vacuolization of plastids— An electron microanalysis study

Chloroplasts in leaf cuttings of Nicotiana rustica L . show swelling of the thylakoid system and formation of plastid vacuoles in the light. These ultrastructural changes may be caused by uptake of K⁺ and Cl- into the aqueous compartments between the thylakoid membranes. Ion changes arid ultrastructural alterations are reversible in darkness.     

Quantitative local x-ray spectral microanalysis of single erythrocytes

Quantitative x-ray microanalysis was used to study the content of sodium, potassium, phosphorus and chlorine in single peripheral red blood cells of man. The method for sample preparation described permits its successful use under clinical and field conditions.     

Quantitative X-ray microanalysis of adrenal medullary cells of young adult and aged rats after glutaraldehyde fixation and potassium dichromate treatment

X-ray microanalysis has been used to detect chromium in the histochemical reaction product resulting from the reaction of noradrenaline with glutaraldehyde during fixation of the rat adrenal medulla and subsequent treatment with potassium dichromate. In unstained ultrathin sections, noradrenaline cells can be identified by their content of highly electron-dense storage granules, which enables individual granules to be analysed quantitatively to assess the amount of bound chromium within them. In young adult (4-month-old) rats the mean chromium content of noradrenaline-containing adrenal medullary granules was 443.6±50.7 mM/kg dry weight. In aged (24-month-old) animals the mean chromium content was 267.0±64.0 mM/kg dry weight which was significantly (P<0.01) lower then the value for the young adult rats. Some noradrenaline cells contained granule populations, which were markedly less electron dense than those in the young adults and this is reflected in the ranges of chromium values recorded between individual cells in the 24-month-old animals. There were also noradrenaline cells in the medulla of the aged animals, which contained highly electron-dense granules but these did not contain as much bound chromium as the highest values recorded in the young adult animals. The results are discussed in the context of the growth of the rat adrenal medulla throughout the lifespan and with respect to the effects of age on the integrity of storage granules.     

Quantitative determination of ferritin by electroimmunoassay

A method for the quantitative determination of tissue ferritin protein is described. It is based on the electroimmunoassay of Laurell [Laurell, C. B. (1966) Anal. Biochem.15, 45–52] and uses the iron content of ferritin for its identification. It measures as little as 0.1 μg of ferritin protein, requires only a few milligrams of tissue, and is rapidly performed.     

Quantitative X-ray microanalysis of halogen elements in biological specimens

Summary Standards were prepared for quantitative X-ray microanalysis of the halogen elements Cl, Br and I in sections of resin embedded biological specimens. Halogenated aromatic compounds were dissolved in resin, which subsequently was polymerized. Homogeneity and stability of the standards were determined and found to be satisfactory. A general procedure of calculation of elemental concentrations according to the continuum method is given for the case that the specimen contains appreciable concentrations of one or more rather heavy elements. It is shown that use of approximations may lead to unacceptable errors, even in the concentration ranges occurring in biological specimens. As a practical application, the concentration of bromine in the chloroplasts of the red alga Chondrus crispus was determined quantitatively. The inner cells contained more bromine than the epidermal cells: in the chloroplasts of the inner cells bromine concentrations of about 6% could be demonstrated.     

Quantitative X-ray microanalysis of halogen elements in biological specimens

Standards were prepared for quantitative X-ray microanalysis of the halogen elements Cl, Br and I in sections of resin embedded biological specimens. Halogenated aromatic compounds were dissolved in resin, which subsequently was polymerized. Homogeneity and stability of the standards were determined and found to be satisfactory. A general procedure of calculation of elemental concentrations according to the continuum method is given for the case that the specimen contains appreciable concentrations of one or more rather heavy elements. It is shown that use of approximations may lead to unacceptable errors, even in the concentration ranges occurring in biological specimens. As a practical application, the concentration of bromine in the chloroplasts of the red alga Chondrus crispus was determined quantitatively. The inner cells contained more bromine than the epidermal cells: in the chloroplasts of the inner cells bromine concentrations of about 6% could be demonstrated.     

Quantitative X-ray microanalysis: Microcrystal standards and excessive background

Ultrathin sections of dentine and CaHPO₄ crystals were measured by X-ray microanalysis in order to test the feasibility of using microcrystal standards for quantitative analysis. Care must be taken if absolute weight percentages are to be calculated after Hall's thin film model, since large uncertainties may arise during determination of background counts, whereas relative weight or atomic ratios provide good results.     

Ultrastructural localization of potassium and calcium in an insect ommatidium as demonstrated by X-ray microanalysis

Summary Ultrastructural localization of potassium and calcium in the ommatidium of the house-cricketGryllus domeslicus L. was studied by X-ray microprobe analysis using samples prepared as thin sections (2 or 5 m) of freeze-dried and embedded tissue. Real resolution was limited by the size of ice crystals (Fig. 2) and estimated as about 1 m.Average values for potassium, calcium, sodium and phosphorus in different cells of the compound eye are given in Table 1.Striking non-uniformity in distribution of these elements over the cells and their compartments was found by probe scanning (Figs. 3, 4, 5). The highest potassium and calcium concentrations were measured in the pigmented zones of photoreceptors and pigment cells. The pigment granules are thought to be the ionic depots of the eye.Potassium and sodium are fully accessible to water in sections of embedded tissue, whereas all the calcium and half of the phosphorus are not.The functional significance of the non-uniformity discovered is briefly discussed.     

Quantitative determination of growth hormone by immunoblotting

Blotting techniques have been extensively used, not only analytically for protein identification, but also preparatively to isolate and purify specific proteins from a large variety of cellular extracts and biological fluids. The process involves the separation of proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transfer of proteins to nitrocellulose membranes, and immunostaining to identify proteins which often are at very low concentrations. Because of the quantitative interactions of the proteins with specific antibodies, we have coupled the immunoblot procedure with photographic and densitometric methods for the quantitative determination of bovine growth hormone (bGH). In this way, the method is suitable for bGH detection and quantitation for a small number of samples by use of a single Western blot analysis. The sensitivity of this method permits determinations of bGH to 0.5 ng. The method uses a comparative procedure in which purified bovine growth hormone is used as a standard.     

Quantitative X-ray microanalysis of spleen lysosomes after acid phosphatase reaction

Summary A quantitative X-ray microanalytical study of 80 spleen lysosomes after histochemical reaction for acid phosphatase was carried out. Lysosomal Fe concentrations showed a skewed distribution, with most lysosomes having relatively low concentrations. The concentration of Pb in the lysosomes (indicative of acid phosphatase activity) showed a close to normal distribution. Although the plot of lysosomal Pb concentrations against Fe concentrations showed a marked scattering of the points, there is a statistically highly significant correlation between accumulated metal and acid phosphatase activity as determined from the Pb concentrations. The relation between concentrations of P and those of Pb in the lysosomes points to the possibility that Pb(H₂PO₄)₂ is the main reaction product of the histochemical acid phosphatase reaction.