Retroaortic left renal vein in testicular cancer patient: potential staging and treatment pitfall.

A 20-year-old man was diagnosed with a left mixed germ cell testicular tumor and clinical staging with computerized tomography suggested left para-aortic subhilar retroperitoneal adenopathy. The patient received 4 cycles of cisplatin, vinblastine and bleomycin chemotherapy but the mass in the left renal hilus area remained unchanged. Subsequent retroperitoneal lymphadenectomy revealed the mass to be a retroaoritc left renal vein type 2. Further confusion occurred during followup in differentiating this anomaly from recurrent neoplasm necessitating evaluation by magnetic resonance imaging. Retroaortic left renal vein represents a potential imaging pitfall in testicular cancer that may facilitate suboptimal staging, treatment and followup.

Adrenal cortical carcinoma with vena cava tumor thrombus requiring cardiopulmonary bypass for resection.

We believe this is the fifteenth case report of adrenal cortical carcinoma with tumor thrombus to the vena cava, and the fourth reported case of a left-side tumor propagating thrombus to the vena cava. The patient underwent successful resection which required cardiopulmonary bypass. The caval tumor thrombus was very friable and gelatinous, unlike many renal cell thrombi, and required special surgical considerations.

Analysis of glycoprotein oligosaccharides using high-pH anion exchange chromatography.

The natural heterogeneity of glycoprotein glycans requires that chromatography be an essential part of structural elucidation. The isomeric nature of oligosaccharide structure requires chromatography which is selective for not only composition, size and anomerity, but also ring substitutions and branching configurations. HPAEC is sensitive to these structural features and thus, has become an important new method for understanding the elusive function of glycoprotein glycans.

Adaptation of a thermospray liquid chromatography/mass spectrometry interface for use with alkaline anion exchange liquid chromatography of carbohydrates.

An interface is described that allows the direct coupling of high-performance alkaline anion exchange liquid chromatography with thermospray mass spectrometry. A membrane suppressor is used to remove nonvolatile alkaline salts from the mobile phase after the chromatographic process is completed and prior to introduction into the mass spectrometer. Examples are given of both isocratic and gradient separations of a three-component test mixture of N-acetylated mono- and disaccharides, followed by on-line mass spectral data acquisition. Sensitivity studies show minimum detection limits for the test compounds to be in the microgram range.

Separation of branched sialylated oligosaccharides using high-pH anion-exchange chromatography with pulsed amperometric detection.

Ten characterized sialylated oligosaccharides from bovine fetuin (B. Bendiak, M. Harris-Brandts, S. W. Michnick, J. P. Carver, and D. A. Cumming, Biochemistry, in press; and D. A. Cumming, C. G. Hellerqvist, M. Harris-Brandts, S. W. Michnick, J. P. Carver, and B. Bendiak, Biochemistry, in press) were chromatographed using high-performance anion-exchange chromatography with pulsed amperometric detection. At near neutral pH values, oligosaccharides were separated according to their number of formal negative charges from sialic acid; however, at alkaline pH, the neutral portion of the oligosaccharides enhanced resolution due to oxyanion formation. Specifically, trisialylated triantennary oligosaccharides containing a Gal-beta(1,3)GlcNAc sequence were more retained and could be completely separated from those having only Gal-beta(1,4)GlcNAc units. Oligosaccharides containing the same number of sialic acids were separated according to the combination of alpha(2,6)- and alpha(2,3)-linked sialic acids (alpha(2,6)-linked sialic acid reduced retention time). The relative molar electrochemical responses for di-, tri-, tetra-, and pentasialylated oligosaccharides were found to be similar (4.8 +/- 14% relative to glucose). Coelution studies were performed with each of the characterized oligosaccharides and the mixture of oligosaccharides which were released from fetuin with N-glycanase. The relative proportion of the major classes of sialylated oligosaccharides (bi-, tri-, tetra-, and penta-) varied significantly in bovine fetuin from different sources.

Separation of fucosylated oligosaccharides using high-pH anion-exchange chromatography with pulsed-amperometric detection.

Fucosylated oligosaccharides of the beta Gal(1----4)GlcNAc-, beta Gal(1----4)Glc-, and beta Gal(1----3)GlcNAc-series were chromatographed on a high-performance anion-exchange pellicular resin under alkaline conditions (pH congruent to 13). Fucosylation of either lactose, lactosamine (Type II chains), or lacto-N-biose (Type I chains) oligosaccharides markedly decreased the retention time (10-38 min) of the non-fucosylated form. The magnitude of the reduction was related to whether fucose replaced Gal [alpha Fuc(1----3)----GlcNAc], whether fucose was alpha-(1----2)-linked to Gal at the end of a chain, or whether fucose was linked in a subterminal position [alpha(1----3) or alpha (1----4)] to Gal or GlcNAc. The results suggest that the decreases in retention times of fucosylated oligosaccharides (10-38 min) is not attributable to the absence of a 6-OH in Fuc but instead to steric and substitution effects which affect the interaction of the most readily ionizable groups of Fuc (2-OH), Gal (2-OH), and GLcNAc (3-OH) with the stationary phase. We show that high-pH anion-exchange chromatography can effectively separate 1----2, 1----3, and 1----4 fucose positional isomers in a single chromatographic step.

High-performance anion-exchange chromatography of oligosaccharides using pellicular resins and pulsed amperometric detection.

High-performance liquid chromatography using pellicular quaternary amine-bonded resins was used to separate a variety of neutral, sialylated, and phosphorylated oligosaccharides. At pH 4.6, sialylated compounds were separated according to number of negative charges, sialic acid linkage [alpha(2,3) compared to alpha(2,6)], and position of sialic acid linkage along a linear saccharide chain. At pH 13, the neutral sugar portion of the sialylated chain had a significant effect on the separation, due to oxyanion formation. Specifically, sialylated tetrasaccharides containing the Gal beta(1,3)GlcNAc sequence were retained much more than their Gal beta(1,4)GlcNAc- or Gal-beta(1,4)GalNAc-sialylated counterparts. Linear phosphorylated oligosaccharides could be completely separated according to number of charges and net carbohydrate content. Partial separation of linear-chain positional isomers, differing in either location of Man-6-PO4 in the chain or linkage position of Man or Man-6-PO4, was accomplished. Branched-chain phosphorylated compounds could be completely separated according to which antennae contained the Man-6-PO4. The electrochemical current generated by oxidation of sialylated, phosphorylated, and neutral oligosaccharides was compared to that of a glucose. The relative molar response factors for neutral, sialylated, and phosphorylated oligosaccharides ranged from 0.2 to 3.2. Neutral oligosaccharides gave the following molar responses for each group of structurally related compounds: (1) mono- and disaccharide, 1-1.3; (2) linear tri- and tetrasaccharides, 1.5-2.0; and (3) branched pentasaccharide-nonasaccharides, 2.4-3.1. Response factors for the sialyated compounds were not as consistent and were affected by linkage position of sialic acid. For oligosaccharides of the same size, increasing phosphorylation resulted in a twofold decrease in response factor for each added phosphate group. Therefore, conversion of sialylated and phosphorylated oligosaccharides to their neutral counterparts, using alkaline phosphatase or neuraminidase, respectively, was required for quantitative analysis of oligosaccharide mixtures using electrochemical response. Using this approach, complete separation of the parent neutral structures was obtained, the relative proportions of the neutral species were quantified, and the amount of sialic acid released was easily determined in a neuraminidase digest.

Separation of positional isomers of oligosaccharides and glycopeptides by high-performance anion-exchange chromatography with pulsed amperometric detection.

High-performance anion-exchange (HPAE) chromatography under alkaline conditions (pH congruent to 13) has been found to efficiently separate neutral oligosaccharides (triose to undecaose) according to molecular size, sugar composition, and linkage of monosaccharide units. The method was able to resolve 1----3, 1----4, and 1----6 positional isomers of neutral oligosaccharides, which are defined as having the same number, type, sequence, and anomeric configurations of monosaccharides but differing in the linkage position of a single sugar. From correlating structural features of different oligosaccharides and retention times, we deduced that at least two factors are operative to determine the superior resolution of oligosaccharides by this type of chromatography: (i) the relative acidities of the hydroxyl groups and (ii) the accessibility of oxyanions of the oligosaccharides to the functional groups of the stationary phase. Splitting of peaks attributable to mutarotation was not observed. Reducing oligosaccharides were much more retained than their reduced counterparts. Linkage of Fuc(alpha 1-3) to GlcNAc of oligosaccharides markedly decreased retention times. Positional isomers of two branched monosaccharides, which differed by 1----6 and 1----4 linkages, were widely separated. The separation of 1----3 and 1----4 positional isomers of both tetrasaccharides and glycopeptides containing undecasaccharides demonstrated the significant improvement in resolution of HPAE compared to previous chromatographic methods by either reverse-phase or amine-bonded stationary phases. Picomole quantities of underivatized oligosaccharides have been detected by triple-pulse amperometric detection, which produced similar responses for a wide range of structures. Quantification of two triantennary glycopeptides from bovine fetuin by using either detector response or 1H NMR was comparable. The N-glycanase-catalyzed release of two 1----4 and 1----3 positional isomers of an undecasaccharide from a tryptic glycopeptide of bovine fetuin could be observed and quantified by direct injection of the enzyme mixture into the chromatograph.

Assessment of glycosylation-site heterogeneity using plasma desorption mass spectrometry.

Plasma desorption mass spectrometry (PD-MS) was used to assess the molecular weight heterogeneity of glycopeptides (6-12 amino acids) from each of the three N-linked glycosylation sites of bovine fetuin (R.G. Spiro (1962) J. Biol. Chem. 237, 382-388). The glycopeptides were purified by a combination of anion exchange chromatography and reverse-phase HPLC. Since no detectable fragmentation was observed in the PD-MS of these asialoglycopeptides, the observation of multiple molecular ions could be attributed to either carbohydrate or peptide heterogeneity. Assignment of molecular ions, within 3 to 5 amu of the theoretical mass, of glycopeptides from each glycosylation site was made from amino acid composition, peptide sequence around the glycosylation sites, and previously reported triantennary oligosaccharide structures (B. Nilsson, N.E. Nordén, and S. Svensson (1979) J. Biol. Chem. 254, 4545-4553). Ion groups differing in mass by one N-acetyllactosamine unit were observed in glycopeptides from the Asn-Asp and Asn-Cys sites, localizing these previously observed biantennary oligosaccharide structures (R.R. Townsend, M.R. Hardy, T.C. Wong, and Y.C. Lee (1986) Biochemistry 25, 5716-5725; S. Takasaki and A. Kobata (1986) Biochemistry 25, 5709-5715) to these two sites. The presence of biantennary oligosaccharides at the Asn-Asp sites could be substantiated using 1H NMR but were not detected in the Asn-Cys glycopeptides. PD-MS was also implemented in the purification protocol for these glycopeptides and proved to be useful in assessing purity of chromatographic fractions which were mixtures of glycopeptides displaying both carbohydrate and peptide heterogeneity. A preparation scheme was developed to obtain molecular ions of desialylated glycopeptides by PD-MS.

Monosaccharide analysis of glycoconjugates by anion exchange chromatography with pulsed amperometric detection.

The method of anion exchange chromatography followed by pulsed amperometric detection (AE-PAD; Johnson, D. C., and Polta, T. Z. (1986) Chromatogr. Forum 1, 37-44) has been applied to the compositional analysis of glycoconjugates. Using 22 mM NaOH as a column effluent, underivatized fucose, galactosamine, glucosamine, galactose, glucose, and mannose were readily separated in 15 min at a flow rate of 1 ml/min. The limit of quantification of the monosaccharides was better than 100 pmol (signal to noise ratio 184:1). AE-PAD was employed to quantify the monosaccharides of several glycoproteins, glycopeptides, and oligosaccharides after hydrolysis with 2 M trifluoroacetic acid. Both neutral and amino sugars could be rapidly estimated in a single chromatographic step using AE-PAD. Complete release of N-acetylglucosamine required more vigorous hydrolysis conditions (Lee, Y. C. (1972) in Methods in Enzymology (Ginsburg, V., Ed.), Vol. 28, pp. 63-73, Academic Press, New York). In both glycopeptides and oligosaccharides, approximately one less residue of Man than predicted was determined. Both AE-PAD and liquid chromatographic analysis of borate-monosaccharide complexes with fluorometric detection (Mikami, H., and Ishida, Y. (1983) Bunseki Kagaku 32, E207-E210) gave similar quantification of mannose and other sugars. The capability of rapid, sensitive quantification of underivitized monosaccharides should facilitate structural analysis of glycoconjugates.

Silent renal obstruction with severe functional loss after extracorporeal shock wave lithotripsy: a report of 2 cases.

We report 2 cases of irreversible renal damage after extracorporeal shock wave lithotripsy. The patients experienced little or no discomfort after lithotripsy and they did not return for followup until 5 months later. One patient had total loss of function of the treated kidney and the other had only 11 per cent remaining function. Both patients had ureteral obstruction documented by retrograde pyeloureterography and neither exhibited any renal arterial pathological condition on selective angiography.

Binding of N-linked bovine fetuin glycopeptides to isolated rabbit hepatocytes: Gal/GalNAc hepatic lectin discrimination between Gal beta(1,4)GlcNAc and Gal beta(1,3)GlcNAc in a triantennary structure.

Glycopeptides were isolated from bovine fetuin after digestion with Pronase, aminopeptidase M, and carboxypeptidase Y. The glycopeptides were derivatized with tert-butyloxycarbonyltyrosine and separated on the basis of peptide by using reverse-phase high-performance liquid chromatography. Using 400-MHz 1H NMR, the asialotriantennary oligosaccharides at each of the three N-linked glycosylation sites were found to be combinations of the following two structures in which the third branch is either Gal beta(1,4)GlcNAc or Gal beta(1,3)GlcNAc: (formula; see text) The asialotriantennary glycopeptides containing all beta(1,4)-lactosamine as the branches were designated Gal beta(1,4)GlcNAc-TRI while triantennary glycopeptides containing beta(1,3)-lactosamine as branch III were termed Gal beta(1,3)GlcNAc-TRI. The Gal beta(1,3)GlcNAc unit was localized predominantly to the branch III arm on the basis of a downfield shift (-0.027 ppm) in the H-1 and upfield shift (0.01 ppm) in the NAc methyl signals from the branch III GlcNAc resulting from Gal beta(1,3) instead of Gal beta(1,4) substitution. Revised assignments are proposed for the H-1's of Gal residues 6 (delta 4.464) and 8 (delta 4.471) [Vliegenthart, J. F. G., Dorland, L., & van Halbeek, H. (1983) Adv. Carbohydr. Chem. Biochem. 41, 209-373] in a Gal beta(1,4)GlcNAc-TRI. The proportion of Gal beta(1,3)GlcNAc-TRI glycopeptides from the Asn-Asp, Asn-Gly, and Asn-Cys sites was found to be 40%, 60%, and 20%, respectively. Analysis of the binding of these glycopeptides, containing from 20% to 60% Gal beta(1,3)GlcNAc as branch III, to rabbit hepatocytes revealed that the greater the proportion of Gal beta(1,3)GlcNAc, the lower the affinity of the mixture. The Kd for Gal beta(1,4)GlcNAc-TRI was found to be between 3.6 and 5.4 nM (P = 0.10) with a mean of 4.4 nM from binding data analyzed by using the LIGAND program [Munson, P. J., & Rodbard, D. (1980) Anal. Biochem. 107, 220-239] and computer simulations of the binding of two ligands as a mixture to one receptor site. The Kd of Gal beta(1,3)GlcNAc-TRI oligosaccharide, prepared by hydrazinolysis, was found to be 305 nM from inhibition studies.

Different modes of ligand binding to the hepatic galactose/N-acetylgalactosamine lectin on the surface of rabbit hepatocytes.

A study of the binding of three different 125I-labeled, galactose-terminated ligands to the hepatic galactose/N-acetylgalactosamine-specific lectin found on the surface of rabbit hepatocytes revealed that the different ligands manifest different physical parameters of binding. Asialoorosomucoid (125I-ASOR) binding was best described as involving two independent classes of binding sites on rabbit hepatocytes, with 161 000 sites/cell with a dissociation constant of 0.44 nM and 292 000 sites/cell with a Kd of 9.7 nM. Asialotriantennary glycopeptide purified from human alpha-1 protease inhibitor and modified with tyrosine at the N-terminus to permit radioiodination (TRI) [Lee, Y. C., Townsend, R. R., Hardy, M. R., Lönngren, J., Arnarp, J., Haraldsson, M., & Lönn, H. (1983) J. Biol. Chem. 258, 199-202] was also found to bind to two apparent classes of binding sites but with different binding parameters: 292 000 sites/cell of Kd = 1.47 nM and 982 000 sites/cell of Kd = 25.3 nM. A synthetic ligand, alpha,beta-diaspartamide of tris[(beta-lactosyloxy)methyl](6-aminohexanamido)methane (di-tris-lac) containing six nonreducing galactose residues [Lee, R. T., Lin, P., & Lee, Y. C. (1984) Biochemistry 23, 4255-4261], was found to bind to 817 000 sites/cell of Kd = 0.63 nM and 1.23 X 10(6) sites/cell of Kd = 25.3 nM. Thus, there were many more total binding sites for TRI or di-tris-lac on the surface of rabbit hepatocytes than there were for asialoorosomucoid, although the dissociation constants were similar for all three ligands.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding of synthetic oligosaccharides to the hepatic Gal/GalNAc lectin. Dependence on fine structural features.

A series of synthetic oligosaccharides, resembling natural N-acetyllactosamine type glycans, were tested for their ability to inhibit the binding of labeled ligand to the mammalian hepatic lectin on rabbit hepatocytes at 2 degrees C. A dramatic hierarchy of inhibitory potency (tetraantennary greater than triantennary much greater than biantennary much greater than monoantennary) could be demonstrated. The range of concentration required for 50% inhibition of labeled ligand binding extended from approximately 1 mM, for the monoantennary oligosaccharides, to approximately 1 nM for triantennary oligosaccharides, even though the absolute Gal concentration increased only 3-fold. It was found that the number of Gal residues/cluster and their branching mode are major determinants of binding affinity of ligands to the hepatic lectin on the surface of hepatocytes.