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A fluorocarbon emulsion (Fluosol DA 20%) was experimented as a blood substitute. Fourty-five rats weighing 320 +/- 30 g. underwent different degrees of hemodilution, by isovolemic exanguino-transfusions. A first group (15 rats) had maximal exchange transfusion (Ht = 0,5%) with Fluosol DA 20% and was followed for 12 hours. A second group (15 rats) (control group) underwent the same procedure, but hydrolyzed gelatin 3% (Emagel) was used instead of Fluosol DA 20%. A third group (15 rats) had partial exchange-transfusion (Ht = 23 +/- 2%) with Fluosol DA 20%, and was followed for 2 months. Cerebral and cardiovascular activity, respiratory exchanges and acid-base balance as well as other basic biological parameters were evaluated in the first and second group. The initial signs of altered cerebral activity were observed with a Ht = 2%, while death occurred at Ht = 0,5% due to respiratory arrest. In the third group, a retention of fluorocarbon particles was demonstrated by gas chromatography in all the organs examined. In this group, lung and spleen lesions were also observed by light microscopy and transmission electron microscopy.

Micro cation exchange chromatography determination of HbA1c does not provide a complete picture of Hb glycation, for it does not determine all the glycated forms of hemoglobin. For the determination of total glycation, we describe here a rod IEF method, which allows the simultaneous quantitation of glycation on alpha and beta globin chains. The method exhibits good sensitivity; it is not affected by artifacts deriving from temperature, hypertriglyceridemia, Hb variants or labile HbA1 (aldiminic Hb). The results obtained indicate that in a normal population approximately 18% of the beta chain and 8% of the alpha chain are glycated. These mean percentages increase in the diabetic to 28% and 12%, respectively. The beta chain is glycated on both valine and lysine residues, while the alpha chain is glycated only on the latter. HbA1 values from micro cation exchange chromatography are significantly related to both alpha and beta glycation. Thus, valinic or lysinic glycation have roughly the same clinical significance.

Differences in the local structure of the heme in the isolated alpha-, beta- and gamma-chains of the adult and fetal human hemoglobin are detected by XANES (X-ray absorption near-edge structure) spectroscopy. The ligand bonding angle to the iron ion in the ligated forms and the displacement of the Fe respect to the porphyrin plane in the deoxy forms are found to be different for each chain.

The body composition of lean control subjects was compared with that of subjects following biliopancreatic diversion for morbid obesity, when their excess weight had been stabilized at under 40 per cent of the ideal body weight value for over two years. Lean body mass, body cell mass and fat were indirectly calculated from total body water and total body sodium, measured by isotope dilution technique. The body composition in the two groups of subjects was essentially normal, as was indicated by the percentage values of the body composition parameters and by the linear correlations between body weight and the lean body compartments size.

The globins from all fast haemoglobin (Hb) components obtainable by Bio-Rex 70 cation-exchange chromatography were examined by isoelectrofocusing on polyacrylamide gel rods with 8.0 mol/l urea. From this analysis HbA1a1 and HbA1a2 seem to be very heterogeneous components. HbA1b is separable into two components, one of which is varied in both the beta chains. Between HbA1b2 and the well-known HbA1c components two chromatographic peaks are separated, one with a noticeable percentage of glucosylated beta chain and one that probably contains HbF. HbA1c has both beta chains glucosylated, while HbA1x seems to be a beta monoglucosylated Hb form. Finally, the early part of the HbAo peak has a large amount of glucosylation on both alpha and beta chains.

A purified and lyophilized sponge of native bovine tendon collagen (Gelfix) was used to plug gastric, jejunal and colonic surgical holes (phi = 6 mm) on 78 white rats. In a control group (18 rats) colonic holes were plugged with autologous epiploon. The mortality rate due to generalized peritonitis, was higher (Fischer test = 4%) in the control group than in animals treated with the heterologous collagen. The healing process was evaluated by light microscopy at sequential intervals ranging from 4 to 120 days after implantation. The heterologous collagen biological activity was evaluated as follows: 1. The sponge was labelled with Tyrosine 125I or mixed with Collagen Methyl 14C methylated. Then the radioactivity of the isolated granulation tissue cells was evaluated. The electrophoresis of these lysed cells, as well as histoautoradiography were performed. 2. Indirect immunofluorescence studies employing rabbit antibodies against fibronectin were also carried out. The heterologous collagen seemed to act as a mechanical support and as a stimulus to chemotactic migration of fibroblasts. It also appeared to participate as a nutrient substitute in the metabolic activity of the granulation tissue. Finally, heterologous collagen fibers were found to be massively bound by fibronectin.

Protein malnutrition is the main early problem with biliopancreatic diversion; gastric volume greatly influences the incidence of protein malnutrition.

The use of reversed-phase high-performance liquid chromatography (RP-HPLC) for the determination of protein sequences is reported. Topics considered include the peptide separation of endoprotease digestion mixtures, the application of HPLC peptide mapping as an efficient system to check the accuracy of an assumed protein sequence, obtained indirectly by DNA sequencing and the use of HPLC for amino acid analysis in the Edman sequence strategy. The use of RP-HPLC for an unconventional sequence strategy is demonstrated; HPLC exopeptidase mapping appears to be particularly useful as a future technique for small terminal sequence analysis. Finally, the coupling of HPLC with fast atom bombardment mass spectrometry is discussed.

Differential binding curve measurements of carbon monoxide and oxygen binding to human hemoglobin F0 under near-physiological conditions (0.1 M NaCl and 15 mM 2,3-diphosphoglyceric acid, pH 7.35, and 37 degrees C) have allowed a detailed description of the binding and linkage between these two gaseous ligands. Comparison with human hemoglobin A0 under identical solution conditions shows that fetal hemoglobin F0 binds oxygen and carbon monoxide with higher affinity than human hemoglobin A0, but with the same cooperativity. Construction of the partition coefficient surface for carbon monoxide and oxygen binding reveals a failure of Haldane's laws for both hemoglobins. Linkage graphs are used to explore the phenomenological properties of the system. The graphs provide a quantitative description of the mechanism of carbon monoxide toxicity on oxygen transport by hemoglobin in vivo and demonstrate striking similarities between the functional properties of fetal and adult hemoglobins.

The effect of inositol hexakisphosphate (IHP) on the spectroscopic (EPR and absorbance) properties of the nitric oxide derivative of ferrous naturally glycated human hemoglobin HbA1c (HbA1cNO) has been investigated quantitatively. The results obtained show that 1) both in the absence and presence of IHP, the EPR and absorbance spectra of HbA1cNO show the same basic characteristics described for the nitrosyl derivative of ferrous HbA0, the nonglycated major component of human hemoglobin (HbA0NO); and 2) HbA1cNO binds IHP with an apparent dissociation equilibrium constant (upsilon = 1.8 x 10(-2) M), which is at least four orders of magnitude higher than that estimated for the polyphosphate interaction with HbA0NO (less than or equal to 3 x 10(-6) M). These data provide further independent evidence that interaction(s) of polyphosphates at the specific cleft between beta-chains along the dyad-axis is sterically hindered in HbA1c by the presence of the two glucose residues covalently bound to the N-termini of beta-chains, this finding being in agreement with the reduced effect of polyanions on HbA1c spectral and ligand-binding properties.

The degree of hepatic steatosis and percentage total nitrogen content were evaluated simultaneously in a group of obese patients and after bypass surgery for obesity. The straight line relationship between sample weight and gas chromatographic response integrals shows that samples had the same water content. The higher concentration of total nitrogen in patients with higher levels of steatosis and the inverse correlation between degree of adipose infiltration and degree of total nitrogen, when water content is the same, suggest that the total liver parenchyma nitrogen concentration closely reflects the structural protein content.

Fourteen isolates belonging to Rhizopus microsporus, Rh. rhizopodiformis, the progeny of Rh. microsporus x Rh. rhizopodiformis and an intermediate species, Rh. chinensis, were serologically tested either by a modification of the exoantigen technique of Kaufman & Standard (1978) or by the more analytical cross immunoelectrophoresis technique with intermediate gel. Common antigenic determinants in the isolates studied indicated that the species, their progeny and the intermediate species Rh. chinensis, are antigenically very closely related. Through absorptions, a specific reference antiserum for Rh. microsporus was obtained. With this adsorbed monospecific antiserum, using the exoantigen test, the identity of the isolates in study, determined after their general morphology as Rh. microsporus, was confirmed. For comparative purposes, the use of reversed phase high performance liquid chromatography for the investigation of fungal isolates is also reported.

The use of Bio-Rex 70 cation-exchange resin for chromatography of normal and diabetic hemoglobin provides a reproducible pattern of the "fast components". Particular attention to the choice of sample preparation, pH of elution, and the increase of ionic strength by sodium chloride linear gradients results in the separation of Hb-A1b into two components and in the isolation of a new component eluting between Hb-A1c and Hb-A0. Experiments with [3H]glucose and the colorimetric test (thiobarbituric acid) normally used to determine the extent of non-enzymatic glycosylation, as well as an increase of this component in diabetic samples compared with normoglycemic ones and a significant linear correlation with Hb-A1c, indicate that this component should be a part of the hemoglobins glucosylated on the epsilon-NH2 group of the lysines of both chains and/or the hemoglobin glucosylated on the alpha-NH2 of the valine of the alpha-chain. We propose to call this component Hb-A1x, pending confirmation of its identity. Normally Hb-A1x accounts for about 3% of Hb-A, but up to 5-7% of glucosylated hemoglobins should be confined to the early part of Hb-A0. In diabetics, the percentage of Hb-A1x rises to 4-5% and that of the other glucosylated hemoglobins increases to 12-16%.

At present, the brachytherapy dosimetry requires the highest level of accuracy in determining the physical parameters of many different radiation sources. Brachytherapy sources differ not only in the nucleus (widely ranging between 30 keV and 1 MeV), but also in the strength and geometrical and filtration characteristics. This work reports the brachytherapy dosimetry carried out in a rigid perspex phantom which allows measurements by means of solid state dosimeters (TLD, semiconductor), ionization chamber and film. Film densities were measured by means of a computerized Perkin-Elmer 1010G microdensitometer, with a spatial resolution of 50 microns. The isodensity lines were dose calibrated by TLD measurements. The dosimetry of the 125I, 192Ir and 198Au sources is reported. The dose distributions of the low energy 125I source, in two different tissues (muscle and adipose) were studied, by means of laboratory built tissue-equivalent sections of the phantoms. The experimental data suggest that this dosimetric procedure is suitable for intercomparison measurements between different radiotherapy centers using the same nuclides in different sources.

The thermodynamic and kinetic properties of the most abundant glycated hemoglobin in human blood, HbA1c, have been studied in detail. They display significant differences as compared to normal hemoglobin, HbA0, in that (1) the shape of the oxygen binding curve of HbA1c in the Hill plot is markedly asymmetrical, with a lower asymptote extending up to approximately 40% oxygen saturation, and the oxygen affinity of the T state being tenfold higher than in HbA0; (2) oxygen pulse experiments on HbA1c show a slower rate of ligand dissociation (k = 25 s-1) even at low levels of oxygen saturation, where the T state is largely predominant; (3) kinetics of CO combination to deoxy HbA1c followed by means of stopped-flow experiments reveal the presence of a quickly reacting component, whose fraction increases upon dilution of hemoglobin. These results show that in contrast to what has been stated by other authors, HbA1c displays functional properties markedly different from HbA0. Analysis indicates that glycation of human hemoglobin affects the T quaternary structure, bringing about a more "relaxed" T state and leading to preferential binding to one type of chain (which is unaffected by chloride ions).

The microchromatographic determination of "fast haemoglobins" is currently used in long-term control of diabetes: its use is made easier by commercially available kits, that are impaired by some analytical variables. For a good standardization of these kits a comparison with an accurate method of analysis is necessary. The macrochromatographic column allows the determination of each fast component with a good precision, and reveals the great complexity of human haemoglobin. Comparison of the two methods shows that the most appropriate temperature of elution for the kit is 20 degrees C and that the increase in the fraction of "fast haemoglobins" in diabetic samples is only related to the HbA1c component.

The X-ray absorption near edge structure (XANES) spectra of the human adult and foetal hemoglobin, of the isolated alpha and beta chains, in the oxygenated forms, and of the oxymyoglobin and carp oxyhemoglobin have been measured at the wiggler beam line of the Frascati Synchrotron radiation facility. The bonding angle of oxygen molecule at the iron site in these hemoproteins in solution, has been measured using the multiple scattering theory for data analysis.

Binding of ADP to platelets enhances the binding of fibrinogen to Gp IIb-IIIa, the specific platelet receptor for adhesive proteins. The linkage between ADP and fibrinogen binding is indirect since ADP does not bind to the same receptor as fibrinogen. We have recently proposed that a third component, once affected by ADP binding, induces a conformational transition of the fibrinogen receptor from the low to the high affinity state, which is responsible for platelet aggregation [De Cristofaro, R., Landolfi, R., Castagnola, M., De Candia, E., Di Cera, E., & Wyman, J. (1988) Proc. Natl. Acad. Sci. USA 85, 8473-8476]. In the present study we provide evidence that this component should be identified with the platelet Na+/H+ antiport. Inhibition of the antiport by pharmacological agents such as amiloride, or else by decreasing extracellular Na+, results in a marked decrease of fibrinogen binding to platelets.

Twenty-four isolates of Penicillium (including a green-spored mutant from a French Brie cheese, Penicillium camemberti) with a proposed relationship to the white cheese mold P. camemberti were investigated by immunological procedures. These penicillia, which are representative of species that have caused considerable taxonomic confusion, had common micromorphology (terverticillate penicilli with rough and smooth stipes and smooth ellipsoidal to subglobose [(3 to 5) X 2 1/2 to 4 1/2 microns] conidia); growth rates; good growth on creatine sucrose agar, cheese, and other products with a high amount of protein and lipid as a primary habitat; production (with the exception of Penicillium solitum) of cyclopiazonic acid; and the ability to grow at low temperatures and water activities. The isolates that were investigated proved to be strictly antigenically related. Absorbed antiserum of the green-spored mutant of P. camemberti showed a specific precipitin band when tested by immunodiffusion either with its homologous reference antigen or with the exoantigens obtained from different isolates. The precipitin band was not present in any P. camemberti starter culture but in many unwanted cheese contaminants. The precipitin band can be used in the purity control of P. camemberti starter culture spore preparations. Analysis of the exoantigens of all the cultures by reversed phase high-performance liquid chromatography allowed us to subdivide these penicillia into nine groups below the species level. The results indicate that P. commune Thom is the wild-type ancestor of P. camemberti.

Killer toxins were isolated from eight selected killer yeasts. Their activity on 100 Candida albicans isolates of human and animal origin was studied. A computer aided system for differentiating C. albicans strains was developed. By using this system, it was possible to differentiate 14 biotypes of C. albicans isolates based on their susceptibility to the killer toxins.

A neutral haemoglobin variant was identified by the combined use of different analytical methods. Isoelectric focusing on immobilized pH gradients (under denaturing conditions) allowed to detect and alpha chain variant. HPLC of tryptic digest showed that its amino-acid variation resided on T alpha 6 peptide (position 41-56). In the fast-atom bombardment (FAB) positive mass spectrum of the tryptic digest, the protonated molecular ion of the T alpha 6 peptide occurred 48 mass units lower than the normal T alpha 6 fragment, corresponding to a Phe----Val substitution. To a partial sequence determination (aminopeptidase digestion of the T alpha 6 peptide, followed by amino acid determination and FAB-MS analysis of the digestion-generated mixture) the substitution appeared to be on Phe43 (CE1). This variant is already known as Hb Torino. The procedure here described proved to be fast and simple, and feasible whenever neutral variants are supposed to occur.

The nutritional status and total body composition of 33 patients with malnutrition after biliopancreatic bypass were studied. Body composition was evaluated directly by measurement of water and total sodium and indirectly by measurement of total body potassium. After nutrition with a parenteral nutritional support the conventional nutritional parameters were restored to the normal values (serum albumin and serum transferrin) and the parameters of body composition were markedly improved even though still altered with respect to healthy controls. Serum albumin and serum transferrin were shown to be very reliable indices of malnutrition but their normalization does not necessarily correspond to optimum body composition and thus to optimal normal nutrition.

Biliopancreatic diversion is a very effective method for weight reduction. In some instances it is too effective and needs to be revised.