J. Howard Bradbury

Determination of the structures of trisaccharides by 13C-n.m.r. spectroscopy

A literature survey of the 13C-n.m.r. chemical-shift data for aqueous solutions of monosaccharides, disaccharides, oligosaccharides, and their methyl derivatives is reported. Analysis of these data reveals a set of empirical rules which may be used in the elucidation of the structure of trisaccharides of known monosaccharide composition, and an example is reported. However, it is not possible to extend the rules to tetrasaccharides and higher saccharides without additional chemical-shift data for related model compounds.

Total cyanide determination of plants and foods using the picrate and acid hydrolysis methods

Abstract A general method has been developed for determination of the total cyanide content of all cyanogenic plants and foods. Ten cyanogenic substrates (cassava, flax seed, sorghum and giant taro leaves, stones of peach, plum, nectarine and apricot, apple seeds and bamboo shoot) were chosen, as well as various model compounds, and the total cyanide contents determined by the acid hydrolysis and picrate kit methods. The hydrolysis of cyanoglucosides in 2 M sulfuric acid at 100 o C in a glass stoppered test tube causes some loss of HCN which is corrected for by extrapolation to zero time. However, using model compounds including replicate analyses on amygdalin, the picrate method is found to be more accurate and reproducible than the acid hydrolysis method. The picrate kit method is available free of charge to workers in developing countries for determination of cyanide in cassava roots and cassava products, flax seed, bamboo shoots and cyanide containing leaves. For eleven different samples of flax seed and flax seed meal the total cyanide content was 140–370 ppm. Bamboo shoots contained up to 1600 ppm total cyanide in the tip reducing to 110 ppm in the base. The total cyanide content of sorghum leaves was 740 ppm 1 week after germination but reduced to 60 ppm 3 weeks later. The acid hydrolysis method is generally applicable to all plants, but is much more difficult to use and is less accurate and reproducible than the picrate method, which is the method of choice for plants of importance for human food.

Picrate paper kits for determination of total cyanogens in cassava roots and all forms of cyanogens in cassava products

The simple semiquantitative picrate method for the determination of total cyanogens in cassava flour has been modified by increasing the concentration of the picrate solution used to make up the picrate papers, such that a linear Beers Law relation between absorbance and cyanogen content is obtained over the range 0–800 mg HCN equivalents kg−1 cassava. The method has been adapted to determine the total cyanogen content of cassava roots and the results compared using the picrate method and the acid hydrolysis method for six different roots from five cultivars. The agreement between the results is satisfactory. The simple method for determination of total cyanogens in cassava roots in the field is available in kit form. The methodology has been modified to allow determination of the three different forms of cyanogens present in cassava flour, viz HCN/CN−, acetone cyanohydrin and linamarin. HCN/CN− is determined by the picrate method in which cassava flour is reacted with 0.1 M sulphuric acid for 3 h at room temperature. HCN/CN− plus acetone cyanohydrin is also determined by the picrate method after treating cassava flour with 4.2 M guanidine hydrochloride at pH 8 for 3 h at room temperature. A comparison has been made of the amounts of the three cyanogens present in six cassava flour samples using the semiquantitative picrate and the acid hydrolysis methods. The agreement between the two methods is satisfactory, which shows that the new methodology works well. The picrate method for determination of the three cyanogens in cassava flour is also available as a kit. © 1999 Society of Chemical Industry

Persistent konzo and cyanogen toxicity from cassava in northern Mozambique

We aimed to detect new cases of konzo and monitor cyanogen exposure from cassava flour in communities previously affected by konzo epidemics in Nampula Province, northern Mozambique. Other objectives were to detect subclinical upper motor neuron damage in schoolchildren and test a new kit to measure urinary thiocyanate concentration. In 1999 and 2000, we carried out active and passive case detection for konzo in Memba and Mogincual Districts. In July and October, 1999, we collected cassava flour from 30 houses in three communities and measured cyanogen concentrations with a picrate kit. In October 1999, we examined all schoolchildren in three communities for ankle clonus and measured urinary thiocyanate concentration in thirty schoolchildren in each of five communities with a picrate kit. We found 27 new cases of konzo in Mogincual District. Mean total cyanogen concentrations in cassava flour varied between both seasons and years, but were always high, ranging from 26 to 186 ppm. Very high mean levels at three sites in November 1998 and July 1999 were probably due to low rainfall in the 1997-1998 season. The proportion of schoolchildren with ankle clonus varied from 8 to 17%. The new picrate kit for urinary thiocyanate worked well; mean concentrations in schoolchildren ranged from 225 to 384 micromol x l(-1). Konzo and sub-clinical upper motor neuron damage persist in poor rural communities in northern Mozambique, associated with high cyanogen concentrations in cassava flour and high urinary thiocyanate concentrations in schoolchildren.

Simple Picrate Paper Kit for Determination of the Cyanogenic Potential of Cassava Flour

A simple picrate paper kit method was developed for the semi- quantitative determination of the cyanogenic potential for cassava Nour. The method involved the addition of linamarase and pH 8 phosphate bu†er (absorbed in a -lter paper disc) to 100 mg of Nour )0E5 ml water placed in a small vial. A rectangle of yellow picrate paper attached to a plastic strip was added and the vial capped and left overnight. The yellow paper changed colour towards brown and its colour was compared with that of a standard colour card with 10 shades of colour which corresponded with cyanogenic potentials of 0E 800 mg HCN equivalents kg~1 Nour (ppm). To obtain a more accurate measure of cyanogenic potential (^20%) the paper was eluted with water and the absorb- ance measured at 510 nm. The cyanogenic potential in ppm was determined from a calibration graph. The methodology is available in kit form. The simple method may be used in the -eld by a relatively unskilled person. The more accurate method requires a spectrophotometer and is suitable for use in simple laboratories in developing countries. 1998 SCI. (

Structure of a marsupial-milk trisaccharide.

Abstract A trisaccharide, which is a major carbohydrate component of the milk of the tammar wallaby and the grey kangaroo, has been identified by chemical, enzymic, g.l.c.-m.s., and n.m.r. methods as O-β- d -galactopyranosyl-(1→3)-O-β- d -galactopyranosyl-(1→4)- d -glucose (3′-galactosyl-lactose).

The acridity of raphides from the edible aroids

The corms and leaves of most cultivars of the edible aroids, the major staple food of about 200 million people in the tropics, are acrid. This means that if eaten raw they cause swelling of the lips, mouth and throat. This effect is related to the presence of needle-like raphides of calcium oxalate. A new method was developed for the separation of raphides using petroleum ether (PE) and then purification by centrifugation. The acrid raphides were shown by X-ray powder diffraction to consist of calcium oxalate monohydrate (whewellite) and their morphology was studied by scanning electron microscopy. Two distinct types of raphides (thick and thin) were observed and druses (rosette like structures of calcium oxalate) were found only in taro leaves. A comparative survey of raphide morphology was made over four species of aroids. The removal of the acridity of raphides by immersion in hydroxylic solvents, but not in PE, indicates the presence of an irritant on the raphides. Acridity is due to the dual action of the sharp raphides in puncturing soft skin and the irritant (a protease) in causing swelling and soreness.

Combination of cassava flour cyanide and urinary thiocyanate measurements of school children in Mozambique

The maximum daily cassava flour intake of children may be calculated from determination of the total cyanide content of cassava flour and urinary thiocyanate levels of school children in samples collected at the same time and place. Four sites, two with and two without recent konzo cases, were chosen for study. In two sites with recent konzo cases, 84% and 93% of school children consumed cassava the previous day, and the calculated maximum daily consumption of cassava was over 700 g. In two sites without recent konzo cases, about 50% of school children consumed cassava the previous day and the calculated daily consumption of cassava flour was less than 150 g. By measurements of cyanide in flour and urinary thiocyanate we are therefore able to distinguish between communities whose diet is almost totally reliant on cassava, and who are therefore susceptible to konzo, and those who have a broader diet and are free from konzo. In another calculation it is shown that 4–23% of the essential S-containing amino acids in the cassava flour consumed by children is used up to detoxify and flour cyanide to thiocyanate. This depletion of methionine and cystine may leads to protein deficiency and may contribute to onset of konzo.

1H Nuclear Magnetic Resonance Study of the Histidine Residues of Insulin

Abstract Insulin has proved difficult to study by nuclear magnetic resonance spectroscopy because of its complex aggregation behaviour in solution and its insolubility between pH 4 and 7. Now for the first time it has been possible to assign the 1 H nuclear magnetic resonances of the H-2 histidine protons of residues B5 and B10 of bovine 2 Zn insulin and Zn-free insulin, and the B5 and A8 residues of hagfish insulin. As expected, the addition of Zn to Zn-free insulin causes virtually no change in the chemical shift or the rate of H-D exchange of the H-2 proton of histidine B5, which is not involved in Zn binding in the 2 Zn insulin hexamer. The rate of H-D exchange of the H-2 proton of histidine B10 is decreased markedly on Zn binding at this residue, but the chemical shift of the resonance remains virtually constant owing to the balancing of an upfield ring current shift of the ordered histidine residues by a downfield shift due to electron withdrawal from the ring nitrogen by the Zn binding.

Carbon-13 NMR spectra of tryptophan, tryptophan peptides and of native and denatured proteins

Abstract The assignment of the carbon-13 magnetic resonance spectrum of l -tryptophan in aqueous solution and in trifluoroacetic acid is made by comparison with the spectra of indoles, by double resonance and by deuteration experiments. On the basis of work on small peptides, it is possible to assign all the downfield resonances in denatured proteins to single types of carbon atoms, with the exception of the carbonyl carbons and the histidine Cγ resonance. In the upfield region there are only about eleven resonances which correspond to single types of carbon atoms from eight different amino acid residues. The spectra of a particular protein denatured by heat, 6 M guanidine · HCl or formic acid are all similar. The spectrum of a native protein is much broader than that of the denatured protein due to small differences in chemical shift which result from environmental changes of some carbon atoms, and line broadening due to decreased mobility of carbon atoms.