The influence of macromolecular polymerization of spin-lattice relaxation of aqueous solutions.

The docking or polymerization of globular proteins is demonstrated to cause changes in proton NMR spin-lattice (T1) relaxation times. Studies on solutions of lysozyme, bovine serum albumin, actin, and tubulin are used to demonstrate that two mechanisms account for the observed changes in T1. Polymerization displaces the hydration water sheath surrounding globular proteins in solution that causes an increase in T1. Polymerization also slows the average tumbling rate of the proteins, which typically causes a contrary decrease in T1. The crystallization reaction of lysozyme in sodium chloride solution further demonstrates that the "effective" molecular weight can either decrease or increase T1 depending on how much the protein is slowed. The displacement of hydration water increases T1 because it speeds up the mean motional state of water in the solution. Macromolecular docking typically decreases T1 because it slows the mean motional state of the solute molecules. Cross-relaxation between the proteins and bound water provides the mechanism that allows macromolecular motion to influence the relaxation rate of the solvent. Fast chemical exchange between bound, structured, and bulk water accounts for monoexponential spin-lattice relaxation. Thus the spin-lattice relaxation rate of water in protein solutions is a complex reflection of the motional properties of all the molecules present containing proton magnetic dipoles. It is expected, as a result, that the characteristic relaxation times of tissues will reflect the influence of polymerization changes related to cellular activities.

Molecular masking and unmasking of the paramagnetic effect of iron on the proton spin-lattice (T1) relaxation time in blood and blood clots.

The contribution of hemolysis, proteolysis and the paramagnetic effect of iron on the proton spin-lattice (T1) relaxation time in blood was examined. Hemolysis induced by sonication resulted in a significant (10%) increase in the T1 relaxation time of whole blood. Proteolysis in both sonicated and unsonicated whole blood samples eventually yielded T1 values which correlated well with the relaxation times of free iron in plasma or water at concentrations comparable to the concentration of iron in whole blood. It is concluded that proteolysis allows the iron atom to express its paramagnetic effect on water relaxation by gradually destroying the hydrophobic nature of the pocket in which iron resides on the hemoglobin molecule. The contribution of various blood components to the T1 relaxation of whole blood was also studied. The T1 values for packed erythrocytes, intact whole blood, sonicated whole blood, plasma and serum proved to be significantly different from each other. Serum was found to have a significantly (12%) longer T1 relaxation time than plasma. Packed clotted blood in vitro showed no change in the T1 time for at least 13 days while packed erythrocytes showed a shortening of T1 time after 6-8 days.

Post mortem changes in skeletal muscle proton spin-lattice relaxation times.

A previously published report indicated that there are early post mortem changes in the pulsed NMR proton spin-lattice relaxation time (T1) of skeletal muscle which must be taken into account in in vitro tissue analysis. We re-examined this subject. When T1 measurements were done by allowing the signal intensity to decay over two orders of magnitude from the original intensity, the T1 decay curves showed more than a single exponential relaxation time component as reported earlier. However, when T1 measurements were done by allowing signal intensity to decay to about one order of magnitude from the original intensity only a single exponential relaxation time component was found. We postulate that the latter method gives the average T1 value of the various macroscopic tissue components present and that this method gives a representative T1 value for the entire tissue. Using data obtained in this manner we could not find significant post mortem changes in the muscle T1 relaxation times during the first four hours but found change at a later time.

Immediate response of 5-methoxytryptophol in the circulation to hypoglycaemic stress induced by insulin.

Pineal indoles have been shown to affect the release of anterior pituitary hormones but details of the interrelationships are lacking. Using a new gas chromatography-mass spectrometry (g.c.-m.s.) assay the concentration of 5-methoxytryptophol (ML) was measured in plasma samples obtained from 16 children undergoing investigation of pituitary function for delayed growth. All the children received an insulin tolerance test (ITT) to study their endocrine response to stress. Some children received luteinizing hormone releasing hormone (LH-RH) and/or thyrotrophin releasing hormone (TRH). The change in concentration of ML during an ITT was similar to the change in concentration of blood sugar; a drop at 20 min followed by a rise at 30 min. This was not significantly altered by the administration of LH-RH or TRH, nor was there a different pattern of response in children who were deficient in growth hormone as opposed to those with idiopathic delayed growth. The fall in concentration of ML with stress may mediate the increased secretion of pituitary hormones. Alternatively, the pineal gland may respond directly to insulin.

Concentration of 5-methoxytryptophol in pineal gland and plasma of the rat.

5-Methoxytryptophol, a serotonin metabolite, was measured by gas chromatography-mass spectrometry in pineal glands, plasma and control tissues (cerebral cortex and salivary glands) from male rats kept in a controlled lighting environment. In the pineal gland the level of 5-methoxytryptophol was significantly higher during the dark period than during the light, the absolute levels being an order of magnitude less than those of melatonin. In the plasma, the levels showed a reverse situation with respect to lighting conditions. No correlation was found between the 5-methoxytryptophol levels in plasma and the pineal gland in individual animals. These results suggest that there is no obvious correlation between pineal content and pineal activity. This may be due to a combination of rapid turnover, secretion and/or peripheral conversion of another 5-methoxyindole to 5-methoxytryptophol.

A sensitive and specific assay for 5-methoxytryptophol in plasma.

5-Methoxytryptophol (ML) is found in the pineal gland and is known to have biological activity especially as an antigonadotrophic agent, but methods have been lacking for its measurement in the circulation. A gas chromatography-mass spectrometry assay using a trimethylsilyl derivative has been developed for the routine measurement of ML in plasma. The assay is of great specificity and has a sensitivity of 20 pmol/l. Studies on the levels of pineal indoles in the circulation, however, have been hampered by the possibility that extraneous compounds are being cross-measured. Thus the specificity of the routine assay has been further validated by comparing it with an alternative assay system where all the major parameters were changed, i.e. derivatizing reagent, internal standard and mass number. Results that were obtained using both assay systems were closely comparable.

Changes in the concentration of 5-methoxytryptophol in the circulation at different phases of the human menstrual cycle.

The pineal indole 5-methoxytryptophol (ML) has been shown to have an antigonadal activity when administered to experimental animals, but data on its normal pattern of secretion have been lacking. Using a new gas chromatography-mass spectrometry assay, the concentration of ML at various phases of the human menstrual cycle has been studied. Daily samples were obtained throughout the month from five women with a normal cycle and two women taking an oral contraceptive. In women with a normal cycle levels of ML were found to be significantly lower in the last third of their cycle; this change was not seen in women taking an oral contraceptive who had low levels throughout the month. The changes in concentration of ML did not correlate with the changes in concentration of gonadotrophins.

Massive excretion of 2-oxoglutaric acid and 3-hydroxyisovaleric acid in a patient with a deficiency of 3-methylcrotonyl-CoA carboxylase.

A three-month old child, presenting with a history of feeding problems, suspected respiratory infection and failure to thrive, later developed fits and a profound irreversible metabolic acidosis. Chromatographic investigation of the urine revealed a gross excretion of 2-oxoglutaric and 3-hydroxyisovaleric acids. The identity of these two acids was confirmed by mass spectrometry. Enzyme studies on liver obtained at post-mortem demonstrated a deficiency of 3-methylcrotonyl-CoA:carbon dioxide ligase (ADP) (EC 6.4.1.4).

The occurrence and identification of o-hydroxyhippuric acid (salicyluric acid) in the urine of sick children.

O-Hydroxyhippuric acid has been identified in the urine of a number of sick children who were not receiving salicylate-containing drugs. Characterisation was effected by thin-layer and gas-liquid chromatography and by gas-liquid chromatography-mass spectrometry. An association between gastro-intestinal dysfunction and the excretion of o-hydroxyhippuric acid was observed in approximately 50% of the patients who excreted this substance.