Steffen Petersen

SOLID-STATE 13C AND 31P NMR ANALYSIS OF URINARY STONES

PURPOSE We investigated the applicability of solid-state nuclear magnetic resonance (NMR) spectroscopy to obtain information about the structure and composition of renal calculi. MATERIALS AND METHODS Various types urinary and bladder stones as well as a variety of presumed constituents were investigated using 13C and 31P magic-angle spinning (MAS) solid-state NMR. Different experimental methods were applied to differentiate resonances from crystalline/amorphous (immobile/mobile) as well as protonated/non-protonated moieties. The NMR spectra were analyzed using multiple-component numerical simulations and iterative fitting to identify and quantify the major amorphous or crystalline organic and inorganic components. RESULTS By comparison of the NMR spectra for the various renal calculi with those obtained under similar conditions for various presumed components, it is demonstrated possible to unambiguously distinguish and quantify the major amorphous or crystalline organic and inorganic components. The components are identified in terms of their isotropic and anisotropic chemical shielding parameters, protonation or proximity of protons, and the degree of crystallinity/mobility. For the calculi investigated we have detected and quantified calcium oxalate, uric acid, struvite, and calcium phosphates that closely resemble brushite and calcium hydroxyapatite. CONCLUSIONS Using 13C and 31P MAS NMR spectroscopy we have been able to account for 60 to 85% (by weight) of the constituents in the calculi investigated. The ability to identify and quantify both crystalline and amorphous components makes solid-state NMR an interesting new method for the compositional analysis of renal calculi.

A comparison between stereological estimates of mean nuclear volume and DNA flow cytometry in bladder tumours

The correlation between stereological estimate of mean nuclear volume and DNA‐content was studied in 55 human urinary bladder tumours. The DNA‐content was determined by flow cytometry on isolated nuclei stained with ethidium bromide. Trout erythrocytes were used as a biological internal standard providing an accurate determination of the DNA‐histogram. An unbiased estimate of the mean nuclear volume (V̄v) was obtained after standard formaline fixation, paraffin‐embedding, sectioning and hematoxylin‐eosin staining using the equation . Here I0 is the length of an intercept measured in a random direction through a test point, which hit a nucleus. A highly significant correlation was found between V̄v and mean DNA‐content of nuclei (2p = 0.0004). A highly significant correlation was also found between V̄v and the highest DNA‐content present in tumours having cell populations with different DNA content (2p = 0.0016). The mean nuclear volume and the DNA‐content also correlated well with the pathologic grade. Although significant the correlations were far from perfect, which indicates that DNA content and mean nuclear volume may provide partly independent biological information. The methods provide objective, unbiased and reproducible data which may improve the possibility of grading and predicting the disease course of human urinary bladder tumours.

Dual-parameter flow cytometry of transitional cell carcinomas. Quantitation of DNA content and binding of carbohydrate ligands in cellular subpopulations

Quantitative DNA measurements and estimates of blood group‐related carbohydrate antigen expression have been used as predictive parameters in transitional cell carciomas (Ca). To obtain an accurate quantitative characterization of cellular subpopulations on the basis of these parameters, the authors developed a dual‐parameter flow cytometric method using a fluorescence‐activated cell sorter. With this method single‐cell suspensions from 26 transitional cell carcinomas were analyzed by means of propidium iodide (red fluorescence) as DNA ligand, and peanut agglutinin (PNA), wheat germ agglutinin (WGA), and anti‐blood group A antibody (aBGA) as carbohydrate ligands. The latter ligands were visualized directly or indirectly by FITC (green fluorescence). The carbohydrate ligand binding was correlated to the DNA content of cell populations in the way that aneuploid populations showed a higher PNA binding (P < 0.0002) and a lower WGA (P < 0.01) and aBGA (P < 0.04) binding than did diploid cell populations. The binding of PNA to aneuploid populations could be further increased (P < 0.004) by neuraminidase treatments. Thus, aneuploid cells express both neuraminic acid substituted and unsubstituted PNA receptors. The carbohydrate ligand binding was cell cycle‐dependent, as it was reduced (<0.008) in the G2‐M phase. A low WGA (P < 0.004) or aBGA (P < 0.02) binding was correlated to tissue invasion. Immunohistochemistry with the carbohydrate ligands showed a good correlation between aBGA (P < 0.0005) and PNA (P < 0.004) binding to tumor cells and flow cytometric assay of these, as well as a correlation (P < 0.003) between cellular location of WGA receptors and flow cytometric assay of these. It seems that dual‐parameter flow cytometry represents an important tool in the characterization of bladder tumors.

Identification of Hemoglobin and Two Serine Proteases in Acid Extracts of Calcium Containing Kidney Stones

By stepwise extraction of kidney stones, first with 10% acetic acid then 0.1 M EDTA, three pools of protein material have been obtained. The soluble material from the acid extract showed well defined bands when analyzed by sodium dodecyl sulfate (SDS) gel electrophoresis, and amino acid sequence analysis of peptides identified hemoglobin as a common constituent. From one stone neutrophil elastase has been identified along with another serine protease of unknown origin. The second pool of material which was soluble in EDTA showed a smear on SDS gel electrophoresis while the third pool consisted of the insoluble matrix. The finding of proteolytic enzymes opens up the possibility that limited proteolysis might be involved in the stone forming process.