Harold G. Parkes

Nuclear magnetic resonance spectroscopy to determine the micellar cholesterol in human bile

The cholesterol of gallstones comes from the vesicular rather than the micellar phase of bile. Progress in this field has been limited because conventional analytical methods disturb the distribution of cholesterol between the two phases. The resonance of the cholesterol C6 proton occurs at a chemical shift of 5.4 ppm, to be shown by 2D NMR to be specific for biliary cholesterol, and arises only from the micellar mode. Thus integration of C6 proton resonance peak area provides a direct non‐invasive determination of the cholesterol distribution in human bile.

Oxidative damage to synovial fluid from the inflamed rheumatoid joint detected by 1H NMR spectroscopy

The glycosaminoglycan hyaluronate (hyaluronic acid, hyaluronan) [l] is a linear repeating disaccharide, @-D-glucuronyl-p-D-Nacetylglucosamine, of very high molecular mass (>4 X 106) which is a major component of the proteoglycan aggregates required for the functional integrity of extra-cellular matrices such as articular cartilage [2]. Hyaluronan is continually secreted in its unaggregated form by the type II synoviocytes and is largely responsible for the very high viscosity of kneejoint synovial fluid [3]. It has previously been demonstrated that in patients with rheumatoid arthritis or other inflammatory joint diseases, synovial fluid hyaluronate is depolymerized with a corresponding modification in the viscoelastic properties of synovial fluid and an increase in the synovial level of low-molecular-mass hyaluronate-derived saccharides [4]. This fragmentation is presumed to occur by the action of reactive oxygen radical species (RORS) [5] since (1) hyaluronidase activity is absent from both normal and inflammatory synovial fluid samples [6], and (2) there is currently a large quantity of experimental evidence implicating the involvement of these chemically-reactive oxygen metabolites in the pathogenesis of inflammatory joint diseases [7-91. In addition to the presence of potential oxygen radical generating systems within the joint, this hypothesis is also supported by the demonstration of hyaluronate depolymerization by such systems in vivo as evidenced by decreases in apparent hyaluronate molecular mass and viscometric parameters. We have previously suggested that hypoxicreperfusion injury during perambulatory motion of the inflamed rheumatoid joint is a mechanism which perpetuates the deleterious production of oxygen-derived radical species within the joint, giving rise to the chronicity of the inflammation [9]. Much of the toxicity of RORS to biological systems is attributable to the highly reactive hydroxyl radical (*OH) [lo], the formation of which appears to be mediated by low-molecular-mass iron chelates. In this communication we present evidence for RORS-mediated damage to knee-joint synovial fluid in patients with inflammatory synovitis. Nuclear magnetic resonance (NMR) spectroscopy has been extensively used for investigating the metabolic profile of body fluids [ll], particularly for monitoring the status and levels of low-molecular-mass endogenous components. We have employed high field proton Hahn spin-echo NMR spectroscopy to detect and characterize a series of lowmolecular-mass species derived from the attack of radiolytically-generated RORS on hyaluronate. We have also utilized this technique to assess RORS-mediated oxidative damage to synovial fluid hyaluronate in vivo during hypoxic-reperfusion injury. In addition, the

Isolation, n.m.r. spectra, and X-ray crystal structures of two isomeric N3P3Cl2[O(CH2)3O]2 derivatives. The first example of dispiro/spiro–ansa isomerism in phosphazene chemistry

From the reaction of N3P3Cl6 with 1, 3-dihydroxypropane two isomeric N3P3Cl2[O(CH2)3O]2 derivatives were isolated, whose n.m.r. spectra suggest, and crystal structures prove, that they represent an example of dispiro/spiro–ansa isomerism, the first of its kind in phosphazene chemistry.

Phosphorus–nitrogen compounds. Part 54. The reactions of geminal N3P3(NH2)2Cl4 and of N3P3(NH2)Cl5 with alkoxide ions in alcohols. The geminal P(NH2)2 to non-geminal P(NH2)(OR) rearrangement. The crystal structures of trans-N3P3(NH2)2(OPrn)4, cis-N3P3(NH2)2(OMe)4, and geminal N3P3(NH2)2(OMe)4

Geminal N3P3(NH2)2Cl4 has been allowed to react with alkoxide ions in alcohol to give rearranged cis- and trans-N3P3(NH2)2(OR)4(R = Me, Et, Prn, or Bun), as well as unrearranged geminal N3P3(NH2)2(OR)4(R = Me). The 1H, 13C, and 31P n.m.r. spectra are reported. The crystal structures of trans-N3P3(NH2)2(OPrn)4, geminal and cis-N3P3(NH2)2(OMe)4 are presented, the last two being present in a 1 : 1 ratio in the same unit cell.

ISOLATION, NMR SPECTRA AND X-RAY CRYSTAL STRUCTURES OF TWO ISOMERIC N3P3CL2[O(CH2)3O]2 DERIVATIVES. THE FIRST EXAMPLE OF DISPIRO / SPIRO-ANSA ISOMERISM IN PHOSPHAZENE CHEMISTRY. COMPARISONS OF TRANS-ANNULAR BRIDGED CYCLOPHOSPHAZENES

Abstract From the reaction of N3P3Cl6 with 1,3-dihydroxypropane two isomeric N3P3Cl2[O(CH2)3]2 derivatives were isolated, whose NMR spectra suggest, and crystal structures prove, them to represent an example of dispiro / spiro-ansa isomerism, the first of its kind in phosphazene chemistry. The crystal structures and NMR spectra are discussed. Extension of the work with difunctional reagents is reported.

In vitro determination by 1H-NMR studies that bile with shorter nucleation times contain cholesterol-enriched vesicles

Although biliary vesicles are considered to be the primary source of cholesterol found in cholesterol gallstones, difficulties in quantitatively separating the different cholesterol transport modes in bile still remain. Proton nuclear magnetic resonance spectroscopy (1H-NMR) offers an alternative approach. Investigations were carried out on both model biles and human gallbladder bile samples: (i) to follow the effect of increasing sodium glycocholate concentrations on the 1H-NMR spectra of arachidonic acid rich-phospholipid, and cholesterol-lecithin vesicles, (ii) to compare the concentrations of total phospholipids in bile determined enzymatically with those obtained by integration of the phospholipid choline head group resonance peak, and (iii) to examine the relationship between biliary cholesterol nucleation time (NT) and the areas of the biliary lipid 1H-NMR peaks. It was found that the molecular motions of vesicle phospholipid, as determined by 1H-NMR, were restricted by saturation with cholesterol. In bile from patients with cholesterol gallstones, the reduced NMR fluidity of the phospholipid choline-head group indicated that the proportion of cholesterol-phospholipid vesicles containing more than 50% cholesterol, on a molar basis, was increased. The ratios of the N+(CH3)3 and = CH proton resonance peaks showed no overlap between samples with cholesterol gallstones and shorter NT and those with either no gallstones or pigment stones and longer NT. 1H-NMR spectroscopy indicates in a non-invasive manner those biles which are prone to cholesterol crystal formation.

THE STEREOCHEMISTRY OF THE NITROGEN ATOM, THE LENGTH OF THE PHOSPHORUS-NITROGEN BOND AND THEIR RELATIONSHIP TO 3 J(PNCC) SPIN-SPIN COUPLING CONSTANTS. THE CRYSTAL STRUCTURES OF N3P3[NMe(CH2)3NMe]Cl4, N4P4(NHEt)6(NEt), AND N4P4(NMe2)5(NHEt)(NEt)

Abstract X-ray crystallographic and 13C NMR spectroscopic investigations demonstrate that 3 J(PNCC) spin-spin coupling constants are reduced when the nitrogen atoms deviate from planarity and the P[sbnd]N bond lengths increase.

THE REACTIONS OF GEMINAL N3P3Ph2Cl4 AND N3P3(NHBut)2Cl4 WITH DIFUNCTIONAL REAGENTS. X-RAY CRYSTALLOGRAPHY, NMR SPECTROSCOPY AND BASICITY OF THE PRODUCTS

Abstract The reactions of geminal N3P3Ph2Cl4 and N3P3(NHBut)2Cl4 with difunctional reagents have been investigated. The N.M.R. spectra of the derivatives are reported. The basicity of these products in nitrobenzene solution is reported and the basicity substituent constants evaluated. X-ray crystallographic data are presented.

THE REACTIONS OF N3P3CL6 AND RELATED COMPOUNDS WITH DIFUNCTIONAL REAGENTS. COMPARISONS AND CONTRASTS

Abstract The reactions of N3P3Cl6 with a variety of difunctional reagents are discussed. NMR spectroscopic and X-ray crystallographic investigations are presented.

X-RAY CRYSTALLOGRAPHIC INVESTIGATIONS OF SOME SPIRO-DERIVATIVES OF CYCLOTRIPHOSPHAZATRIENE

Abstract The crystal structures of a number of spiro derivatives of cyclotriphosphazatriene are reported. Attempts are made to relate these to NMR and other physical properties.