Ian H. Sadler

Patterns of methyl and O-acetyl esterification in spinach pectins: new complexity.

Driselase-digestion of cell walls from suspension-cultures of spinach (Spinacia oleracea L.), followed by anion-exchange chromatography, gel-permeation chromatography, preparative paper chromatography and preparative paper electrophoresis, yielded ten uronic acid-containing products in addition to free galacturonic acid (GalA). These included 4-O-methylglucuronic acid, alpha-L-rhamnopyranosyl-(1-->4)-D-glucuronic acid and several oligosaccharides containing GalA residues. The structures were unambiguously determined by a combination of 1- and 2-dimensional NMR spectroscopic techniques. Five of the six homogalacturonan-derived oligosaccharides purified contained 3-O-acetyl-GalA residues; however, methyl-esterified GalA residues occurred adjacent to both 2-O-acetyl-GalA and 3-O-acetyl-GalA residues. An acetylated, rhamnogalacturonan-I-derived oligosaccharide that was purified also contained 3-O-acetyl-GalA residues. Taken together with published data, our findings indicate considerable diversity in the patterns of pectin esterification. The implications for the action of pectin esterases are discussed.

The Biosynthetic Incorporation of the Intact Leucine Skeleton into Sterol by the Trypanosomatid Leishmania mexicana

The amino acid leucine is efficiently used by the trypanosomatid Leishmania mexicana for sterol biosynthesis. The incubation of [2-13C]leucine withL. mexicana promastigotes in the presence of ketoconazole gave 14α-methylergosta-8,24(241)-3β-ol as the major sterol, which was shown by mass spectrometry to contain up to six atoms of 13C per molecule. 13C NMR analysis of the 14α-methylergosta-8,24(241)-3β-ol revealed that it was labeled in only six positions: C-2, C-6, C-11, C-12, C-16, and C-23. This established that the leucine skeleton is incorporated intact into the isoprenoid pathway leading to sterol; it is not converted first to acetyl-CoA, as in animals and plants, with utilization of the acetyl-CoA to regenerate 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). An inhibitor of HMG-CoA synthase (L-659,699) blocked the incorporation of [1-14C]acetate into sterol but had no inhibitory effect on [U-14C]leucine incorporation. The HMG-CoA reductase inhibitor lovastatin inhibited promastigote growth and [U-14C]leucine incorporation into sterol. The addition of unlabeled mevalonic acid (MVA) overcame the lovastatin inhibition of growth and also diluted the incorporation of [1-14C]leucine into sterol. These results are compatible with two routes by which the leucine skeleton may enter intact into the isoprenoid pathway. The catabolism of leucine could generate HMG-CoA that is then directly reduced to MVA for incorporation into sterol. Alternatively, a compound produced as an intermediate in leucine breakdown to HMG-CoA (e.g. dimethylcrotonyl-CoA) could be directly reduced to produce an isoprene alcohol followed by phosphorylation to enter the isoprenoid pathway post-MVA.

Biosynthesis of polyketide-terpenoid (meroterpenoid) metabolites andibenin B and andilesin A in Aspergillus variecolor

Incorporation of 13C-labelled acetates and methionine indicate that andibenin B (1) and andilesin A (5), C25 metabolites of Aspergillus variecolor, are biosynthesised via a mixed polyketide-terpenoid pathway. 18O2-Labelling studies, and incorporation of aromatic precursors and mevalonic acids variously labelled with 13C, 2H and 18O provide evidence for the extensive oxidative and other modifications involved in the elaboration of the highly oxygenated polycyclic structures found in these metabolites. The biosynthetic interrelationships among these and other complex meroterpenoid metabolites are discussed.

Biochemical prognostic markers of outcome in non-paracetamol-induced fulminant hepatic failure

Background. Fulminant hepatic failure (FHF) is associated with major metabolic disturbances, the onset and severity of which can predict clinical outcome. This study uses admission blood samples to identify early biochemical markers of clinical outcome in patients with non-paracetamol–induced FHF. Patients and Methods. Fifty-nine patients admitted to the Scottish Liver Transplant Unit with non-paracetamol–induced FHF were studied. Plasma samples were collected at a median of 5.4 hr after admission to our unit and analyzed using conventional laboratory tests and nuclear magnetic resonance spectroscopy. Results. A total of 19 patients underwent transplantation, 15 patients died without undergoing transplantation, and 25 patients survived with medical management alone. There were significantly lower levels of lactate, alanine, valine, and bilirubin and significantly higher levels of pyruvate and albumin in patients who survived spontaneously compared with the other two groups. By use of multiple logistic regression analysis, an equation was devised that best predicted clinical outcome: 0.5×(albumin [g/L])−2×(lactate [mmol/L])−36×(valine [mmol/L])−38×(pyruvate [mmol/L]). Values of less than 2 were associated with poor clinical outcome and had a positive predictive value of 91%, a negative predictive value of 86%, a sensitivity of 94%, and a specificity of 86% for death or transplantation. This algorithm can be applied on admission, thus expediting decision-making. Conclusion. We identified biochemical markers that may be useful in predicting outcome in patients with non-paracetamol–induced FHF and should be evaluated further in a different patient population.

3-O-Methyl-d-galactose residues in lycophyte primary cell walls

Acid hydrolysis of cell wall-rich material from young leaves of the lycophyte Selaginella apoda (L.) Spring yielded substantial amounts of 3-O-methyl-D-galactose (1) in addition to the usual major monosaccharides (glucose, galactose, arabinose, xylose and galacturonic acid). The yield of 1 approximately equalled that of galacturonic acid. Compound 1 was identified as 3-O-methylgalactose by its 1H and 13C NMR spectra, and shown to be the D-enantiomer by its susceptibility to D-galactose oxidase. Compound 1 was detected in acid hydrolysates of the alcohol-insoluble residues from young leaves of all lycophytes tested, both homosporous (Lycopodium, Huperzia and Diphasiastrum) and heterosporous (Selaginella). It was not detectable in the charophyte green algae Coleochaete scutata, Chara coralina or Klebsormidium flaccidum, any bryophytes [a hornwort (Anthoceros), four liverworts and three mosses], or any euphyllophytes [a psilopsid (Psilotum), a horsetail (Equisetum), eusporangiate and leptosporangiate ferns, the gymnosperm Gnetum, and diverse angiosperms]. A high content of 1 is thus an autapomorphy of the lycophytes.

A biochemical prognostic model of outcome in paracetamol-induced acute liver injury.

Background. The aim of this study was to develop a prognostic model of outcome for patients with paracetamol induced acute liver injury based on admission parameters Methods. We used a cohort of 97 patients admitted to the Scottish Liver Transplant Unit between 1997 and 1998 to identify biochemical prognostic markers of outcome and thus create a prognostic model. Blood samples were taken on admission for analysis. The model was subsequently validated by testing it on a second cohort of 86 patients admitted between 1999 and 2000. Results. The following were identified as independent variables of poor prognosis (death/ transplant); phenylalanine, pyruvate, alanine, acetate, calcium, haemoglobin and lactate. A prognostic model was then constructed by stepwise forward logistic regression analysis: (400 × Pyruvate mmols/L) + (50 × Phenylalanine (mmols/L) – (4 × Hemoglobin (g/dL). A value of <16 had an accuracy of 93% in predicting death correctly. When applied to the validation cohort this model had a positive predictive value of 91%, a negative predictive value of 94%, a sensitivity of 91%, and a specificity of 94%. On the same population overall, the positive and negative predictive value of the Kings criteria were 94% and 93% respectively, whereas their sensitivity and specificity were 88% and 96% respectively. Conclusions. Using admission characteristics our model is able to identify patients who die from paracetamol overdose fulminant hepatic failure as accurately as Kings College criteria, but at a much earlier stage in their condition.

The simulated microgravity environment maintains key metabolic functions and promotes aggregation of primary porcine hepatocytes

The high aspect ratio vessel allows the culture of primary porcine hepatocytes in an environment of low shear stress and simulated microgravity. Primary porcine hepatocytes have been difficult to maintain in culture long term while preserving their metabolic functions. This study was carried out in order to characterise key metabolic functions of cell aggregates formed by primary porcine hepatocytes cultured in a high aspect ratio vessel for a predetermined period of 21 days. 10(8) porcine hepatocytes were loaded into the high aspect ratio vessel and continuously rotated during the experiments. 0.7 ml of the culture medium was sampled on days 1, 2, 4, 7, 10, 14 and 21. 1H nuclear magnetic resonance spectroscopy of the culture medium, using the presaturation technique, assessed the following: glucose metabolism, glutamine synthesis and ketogenesis. There was glucose breakdown anaerobically during the first 10 days as manifested by lactate production and pyruvate and threonine consumption. After day 10 there was significantly smaller lactate production (day 1 vs day 10 P < 0.01), and significantly smaller pyruvate (day 1 vs day 14 P < 0.03) and threonine consumption (day 1 vs day 10 P < 0.002), indicative of an aerobic metabolic pattern. Significantly more glutamate was produced after day 10 (day 1 vs day 10 P < 0.031), and more glutamine was consumed after day 14. There was a steadily diminishing production of acetate which reached a minimum on day 14 (day 2 vs day 14 P < 0.00014). After an initial 10 day period of acclimatisation cell aggregates formed in the high aspect ratio vessel switched from the anaerobic pattern of metabolism to the more efficient aerobic pattern, which was exhibited until the experiments were terminated. The high aspect ratio vessel is suitable for long-term culture of porcine hepatocytes and it is worthwhile carrying out scale-up feasibility studies.

Characterization of oligosaccharides from the chondroitin/dermatan sulfates. 1H-NMR and 13C-NMR studies of reduced trisaccharides and hexasaccharides.

Chondroitin and dermatan sulfate (CS and DS) chains were isolated from bovine tracheal cartilage and pig intestinal mucosal preparations and fragmented by enzymatic methods. The oligosaccharides studied include a disaccharide and hexasaccharides from chondroitin ABC lyase digestion as well as trisaccharides already present in some commercial preparations. In addition, other trisaccharides were generated from tetrasaccharides by chemical removal of nonreducing terminal residues. Their structures were examined by high‐field 1H and 13C NMR spectroscopy, after reduction using sodium borohydride. The main hexasaccharide isolated from pig intestinal mucosal DS was found to be fully 4‐O‐sulfated and have the structure: ΔUA(β1–3)GalNAc4S(β1–4)l‐IdoA(α1–3)GalNAc4S(β1–4)l‐IdoA(α1–3)GalNAc4S‐ol, whereas one from bovine tracheal cartilage CS comprised only 6‐O‐sulfated residues and had the structure: ΔUA(β1–3)GalNAc6S(β1–4)GlcA(β1–3)GalNAc6S(β1–4)GlcA(β1–3)GalNAc6S‐ol. No oligosaccharide showed any uronic acid 2‐sulfation. One novel disaccharide was examined and found to have the structure: GalNAc6S(β1–4)GlcA‐ol. The trisaccharides isolated from the CS/DS chains were found to have the structures: ΔUA(β1–3)GalNAc4S(β1–4)GlcA‐ol and ΔUA(β1–3)GalNAc6S(β1–4)GlcA‐ol. Such oligosaccharides were found in commercial CS/DS preparations and may derive from endogenous glucuronidase and other enzymatic activity. Chemically generated trisaccharides were confirmed as models of the CS/DS chain caps and included: GalNAc6S(β1–4)GlcA(β1–3)GalNAc4S‐ol and GalNAc6S(β1–4)GlcA(β1–3)GalNAc6S‐ol. The full assignment of all signals in the NMR spectra are given, and these data permit the further characterization of CS/DS chains and their nonreducing capping structures.

Full assignment of the proton and carbon-13 nmr spectra of 2,3,6 -tri-o-methyl-β-cyclodextrin

Abstract The proton and carbon-13 NMR spectra of 2,3,6 -tri-O-methyl-β-cyclodextrin in deuteriochloroform have been fully and unambiguously assigned using homonuclear and selective heteronuclear spin decoupling and two-dimensional homo- and heteronudear correlation NMR spectroscopy. Corrections are made to some earlier literature assignments.

Clerodane diterpenes from Casearia corymbosa stem bark

Abstract Fifteen diterpenes have been isolated from the stem bark of Casearia corymbosa . Nine of these are novel and have been characterized, on the basis of spectral data and comparison with known compounds, as (relative stereochemistry) ent -2β-hydroxy-3,4-dihydro-4α-isozuelanin, ent -3,4-dihydro-4α-isozuelanin-2β-acetate, ent -3,4-dihydro-4α-isozuelanin-2β-(2-methyl)-butyrate, 18-hydroxy-2-oxo-8β,9β,10α-cleroda-3,13(16),14-triene, 18,19α-diacetoxy-2-oxo-8β, 9β,10α-cleroda-3,13(16),14-triene, ent -6β-hydroxyisozuelanin-2β-(2-methyl)butyrate, ent -6β-hydroxyisozuelanin-2β-(2-methyl)propanoate, ent -6β-methoxyisozuelanin-2β-(2-methyl)butyrate, and ent -6β-methoxyisozuelanin-2β-(2-methyl)propanoate. In addition six known diterpenes [ ent -(−)-kaur-16-en-3β-ol, 3α-hydroxy-(−)-13-epimanoyl oxide, (−)-13-epimanoyl oxide, ent -(−)-kaur-16-en-19-oic acid, ent -(−)-kaur-16-en-19-al and ent -(−)-kaur-16-en-19-ol acetate] and the sesquiterpene caryophyllene-4,5-oxide were isolated.