Robert E. Hill

The Biogenetic Anatomy of Vitamin B6 A 13C NMR INVESTIGATION OF THE BIOSYNTHESIS OF PYRIDOXOL IN ESCHERICHIA COLI

It is shown by incorporation experiments with 13C bond-labeled substrates, followed by analysis by means of 13C NMR spectroscopy, that two compounds, 1-deoxy-D-xylulose (12) and 4-hydroxy-L-threonine (13), serve as precursors of pyridoxol (vitamin B6) (1) in Escherichia coli. Together, these two compounds account for the entire C8N skeleton of the vitamin. 1-Deoxy-D-xylulose supplies the intact C5 unit, C-2′,2,3,4,4′ of pyridoxol. 4-Hydroxy-L-threonine undergoes decarboxylation in supplying the intact C3N unit, N-1,C-6,5,5′. Both precursors are ultimately derived from glucose. The C5 unit of pyridoxol that is derived from 1-deoxy-D-xylulose originates by union of a triose phosphate (yielding C-3,4,4′) with pyruvic acid (which decarboxylates to yield C-2′,2). D-Erythroate (11) enters the C3 unit, C-6,5,5′, and is therefore an intermediate on the route from glucose into 4-hydroxy-L-threonine.

Smith-Lemli-Opitz syndrome: phenotypic extreme with minimal clinical findings.

Smith-Lemli-Opitz syndrome (SLO) is caused by inherited enzymatic deficiency of 7-dehydrocholesterol-delta7-reductase and resultant cholesterol deficiency. It comprises a characteristic combination of facial features, malformations, and mental retardation. We report on three related patients (two brothers and their first cousin) with mental retardation and minimal physical signs in whom the diagnosis of SLO was delayed for a number of years. The presence of a third-degree relative in the absence of consanguinity in this family supports the proposed high population carrier frequency. Our report suggests that cases of mild SLO remain undiagnosed and untreated, and that awareness of this common cause of mental retardation is low.

Fumaric aciduria: a new organic aciduria, associated with mental retardation and speech impairment

Two siblings are described who present with fumaric aciduria, a hitherto unreported organic aciduria. The results of our analytical investigations using gas chromatography/mass spectrometry, and the clinical presentation of the patients, are consistent with the notion that the fumaric aciduria is caused by an inherited defect which leads to a net secretion of fumaric acid by the renal tubules.

Growth response to 4-hydroxy-l-threonine of Escherichia coli mutants blocked in vitamin B6 biosynthesis

Mutants of Escherichia coli (pdx B and pdx C) which are blocked in the biosynthesis of pyridoxol (vitamin B6) showed a growth response to 4‐hydroxy‐l‐threonine. This observation constitutes the first direct evidence in support of the view that 4‐hydroxy‐l‐threonine is implicated in the biosynthesis of vitamin B6. 1‐Aminopropan‐2,3‐diol, the decarboxylation product of 4‐hydroxy‐l‐threonine, does not support the growth of these mutants. Deuterium from deuterium‐labelled 1‐aminopropan‐2,3‐diol was not incorporated into pyridoxol.

Biosynthesis of vitamins B1 and B6 in Escherichia coli: concurrent incorporation of 1-deoxy-D-xylulose into thiamin (B1) and pyridoxol (B6)

It is shown by 13C NMR spectroscopy that, in Escherichia coli mutant WG2, the C-2,-3 bond of [2,3-13C2]-1-deoxy-D-xylulose 2 enters C-4,-5 of the thiazole unit of thiamin (B1) 1 and C-2,-3 of pyridoxol (B6) 3, providing the first direct evidence that the intact C5 chain of 1-deoxy-D-xylulose is incorporated concurrently into each of the two B Vitamins.

Mass spectrometry and clinical chemistry

Over the last ten to fifteen years there has been an enormous increase in the sophistication of the analytical instruments used in the clinical chemistry laboratory. That the majority of these instruments is designed for the automated and rapid analysis of body fluids reflects a trend in modern clinical chemistry to invest large amounts of money into capital equipment in order to process large numbers of samples in the least labour-intensive way. Provision of such a service is. however, only one aspect of clinical chemistry. Another responsibility is to provide to smaller patient groups, laboratory services of a more investigative nature which, because of their complexity and limited numbers, do not lend themselves to automation. Also. the discipline and its members should be involved in research into the physiological chemistry of health and disease, and into the application of new methods of analyses to investigate these processes. These responsibilities also require the investment of capital for instrumentation and of time for the investigations. However, since the returns on these investments are different from, and less pecuniary than, those from the automated instrumentation. they are often more difficult to justify. especially in times of fiscal constraint. An example of this type of investment is the use of analytical mass spectrometry in the clinical laboratory. The purpose of this review is to show how this technique has already been successfully adapted to problems in clinical chemistry. On the broader front, it describes one example of how clinical chemistry can carry out the innovative and investigative work alluded to above, which is so crucial to its long-term survival as a scientific discipline. The review will be divided into three sections. The first will describe the types and modes of usage of mass-spectrometer systems that are currently employed in clinical laboratories. The second will review the recent applications of these instruments to clinical chemistry problems. A final. shorter section. will deal with aspects of mass

Biosynthesis of vitamin B6: incorporation of three-carbon units.

Pyridoxol, one of the forms of vitamin B6, is derived from three glycerol units. One of these is incorporated by way of pyruvate as a two-carbon fragment at the oxidation level of acetaldehyde. The other two glycerol units are incorporated intact, possibly by way of triose phosphate.

The widening horizons of bioanalytical mass spectrometry.

Two technological advances of mass spectrometry during the last decade have particular relevance for analytical biochemistry, where the complex matrix of biological fluids challenges the specificity of conventional mass analysis, and where the compounds of greatest interest frequently have a large molecular weight. The first is tandem mass spectrometry (mass spectrometry/mass spectrometry), which emerged around 1980, and has since matured to the point that commercial instruments are now readily available. This method can dramatically enhance the specificity of an analytical procedure, thereby increasing the signal to noise ratio of measurements, and thus indirectly also sensitivity. The second technique is ‘spray ionization’, which was introduced in mid-decade and continues to develop today. The advantage of this latter method is that it allows mass spectrometry to be applied to molecules of high mass and low volatility such as proteins and peptides, oligonucleotides and polysaccharides which previously were not analyzable by mass spectrometry. This brief review outlines these new methods, and gives examples of their use. Their implications for biology and biochemistry are emphasized.

Methods of therapeutic drug analysis.

Chromatographic and homogeneous immunoassay methods are in widespread use in the clinical laboratory. This manuscript reviews the basic principles of each with particular reference to their use in the quantitative assay of therapeutic drugs in serum.

Biosynthesis of vitamin B6. Incorporation of glycolaldehyde into pyridoxal

Radioactivity from [14C]glycolaldehyde enters pyridoxal phosphate specifically, and is confined to the two-carbon unit C(5)–C(5′). Glycolaldehyde enters as an intact two-carbon unit, the aldehyde carbon atom supplying C-5, and the carbinol carbon atom C-5′ of the vitamin. These observations are interpreted in terms of the early stages of the biosynthesis of vitamin B6.