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In order for your submitted work to be considered for assessment you must attend both the introductory talks and the practical demonstration for this experiment - registers will be taken at both.
You will be given a demonstration of the operation of an NMR spectrometer for the analysis of selected bodily fluids. Sorry but we only have one such spectrometer so it has to be a quick demonstration rather than a hands on experience
Following the introductory lectures and the lab demonstration and in conjunction with the additional notes provided on the Blackboard site, produce a written report which covers/answers all of the following.
Describe briefly the major components of a NMR spectrometer and their function.
Figure 1. Components of an NMR spectrometer. (Reed, Holmes, Weyers, & Jones, 2007)
The Magnet - The capability of an NMR instrument is critically dependent upon the magnitude and homogeneity of the static magnetic field and on the bore size of the magnet. There are three main types of magnet; permanent, resistive, and superconducting. (Gadian, 2004)
The Gradient System - The generation of magnetic resonance images relies on the appropriate use of pulsed magnetic field gradients. These gradients are generated in the same way as those produced by the shim coils, i.e. by specially constructed coils mounted within the bore of the magnet, designed to produce field gradients of the required strength and linearity. (Gadian, 2004)
The Transmitter - The transmitter generates radiofrequency pulses of the appropriate frequency, power, shape, and timing. It contains a frequency generator, a waveform generator shape the pulses as required, a 'gate' which switches the transmission on and off at the required times, and a power amplifier which boosts the radiofrequency power to the values that are required in Fourier-transform NMR. (Gadian, 2004)
Figure 2. A simple block diagram of the transmitter. (Gadian, 2004)
The Radiofrequency coil(s) - The Radiofrequency coils are used for transmitting the B1 field into the region of interest, and for detecting the resulting signal. In some cases, the same coil is used for transmission and reception, while in others it may be preferable to use separate transmit and receive coils. (Gadian, 2004)
The Receiver - The design of a modern digital receiver centres around an analog to digital converter (ADC), which samples the analog NMR signal and converts it into digital format. Important characteristics of the ADC are its conversion bandwidth and resolution.
The Computer - The computer has a wide range of function. Its main functions are: (i) to control the radiofrequency and field gradient pulses; (ii) to accumulate the data; and (iii) to process and display the data. (Gadian, 2004)
The magnet produces the Bo field necessary for the NMR experiments. When nuclei interact with a uniform external magnetic field, they behave like tiny compass needles and align themselves in a direction either parallel or anti parallel to the field. The two orientations have different energies, with the parallel direction having a lower energy than the anti parallel.
Immediately within the bore of the magnet are the shim coils for homogenizing the Bo field. Within the shim coils is the probe. The probe contains the Radiofrequency (RF) coils for producing the B1 magnetic field necessary to rotate the spins by 90o or 180o. This will be done by the RF transmitter shown in figure 1. The RF coil also detects the signal from the spins within the sample. These signals will be detected by the RF receiver in figure1. The sample is positioned within the RF coil of the probe. Some probes also contain a set of gradient coils. These coils produce a gradient in Bo along the X, Y, or Z axis.
The heart of the spectrometer is the computer. It controls all of the components of the spectrometer. The RF components under control of the computer are the RF frequency source and pulse programmer. The source produces a sine wave of the desired frequency. The pulse programmer sets the width, and in some cases the shape, of the RF pulses. The RF amplifier increases the pulses power from milli Watts to tens or hundreds of Watts. The computer also controls the gradient pulse programmer which sets the shape and amplitude of gradient fields. The gradient amplifier increases the power of the gradient pulses to a level sufficient to drive the gradient coils.
The operator of the spectrometer gives input to the computer through a console terminal with a mouse and keyboard. Some spectrometers also have a separate small interface for carrying out some of the more routine procedures on the spectrometer. A pulse sequence is selected and customized from the console terminal. The operator can see spectra on a video display located on the console and can make hard copies of spectra using a printer.
Comment on the nature, volume, condition, etc. required of a sample for nmr studies on biofluids.
An important aspect of conducting NMR spectroscopy on biological fluids and tissues is suppression of large interfering resonances, in particular from water, buffers and cosolvents (in the case of extracts). It is also important to be able to apply accurately shaped (non-rectangular) r.f pulses and/or magnetic field gradients across samples to enable diffusion measurements, multidimensional NMR experiments, and the latest solvent suppression approaches. (Gadian, 2004)
In any kind of NMR probe, there are two sample volumes to consider. First is the total volume of sample required (the "sample" volume) and second is the "active volume" or the volume of sample that is exposed to the r.f coils. For probes with the commonly used saddle coil, the ratio of active/sample volume is ~0.5. Typical sample volumes for metabonomics applications range from 120 to 500 µl, a range that is normally adequate for commonly available biofluids such as urine or plasma from anything larger than a mouse. There are also numerous examples of small volume probes (1-30µl) that could have potential uses in certain applications on rare or hard to-obtain biofluids such as CSF or synovial fluids from small laboratory animals. (Gadian, 2004)
No pre-treatment of the sample is required. The metaobiltes which are present in sufficiently mobile form and at sufficient concentration to give detectable signals. For in vivo studies a minimum concentration of 0.2mM is normally required. The amount of sample to be analysed itself is limited by instrument/magnet design but for simple solution studies a typical maximum volume is 0.5 -1 ml. For the less sensitive elements therefore it is desirable to have more concentrated solutions
Hydrogen NMR spectra can be obtained in less than one minute depending on concentration of analytes in sample. 8 combined 'scans' (each of 1-2 seconds duration) is usually enough to give a clear signal. Other nuclei are less sensitive and require more combined scans eg 13C can require a few hours of repeated scanning before signals are clear.
Comment on technical aspects such solvent interferences, exchangeable Hydrogens,experiment duration, etc that are specific/relevant to NMR of biofluids.
The presence of a water (HDO) peak will only serve to degrade the quality of NMR spectra.
The concentration of water in an aqueous solution is about 55M and therefore the signal from water itself usually dwarfs/masks weaker signals. in a normal spectrum but a technique of 'water-suppression' is commonly used to reduce the dominance of this peak and protons in the sample that exchange with water.
In order to remove any interferences from solvent signals during NMR analysis, solvent suppression techniques are employed, the main ones being presaturation and WET (Water suppression Enhanced through T1 effects). The former is a long-standing method that uses shaped pulses to saturate the solvent resonance(s). The WET method uses selective pulses to excite the solvent resonances then dephasing gradient pulses to destroy them. The two techniques take 0.5-2 s and 50-100 ms, respectively, so the WET method is preferred for continuous-flow NMR.
The time to acquire a spectrum depends most critically the number of accumulated scans and hence on the sensitivity of the nucleus under investigation and correspondingly the concentration of the sample.
In general, as molecules become increasingly immobilized they produce broader signals. Therefore spectra of living systems revel narrow signals from metabolites which have a high degree of molecular mobility, whereas macromolecules, which are highly immobilized (such as DNA and membrane phospholipids), produce very much broader signals.1 H NMR spectroscopy imposes particularly stringent requirements. High field spectrometers that are used for studies of solutions may have field homogeneity as 1 part in 109, although of course this is over a much smaller sample volume (e.g. 0.5ml) than the volumes characteristic of in vivo studies. Much better spectral resolution can be achieved using high field system study relatively small volumes of body fluids or of cell or tissue extracts. A great deal of information can be derived from such studies. (Gadian, 2004)
The poor sensitivity of NMR imposes limitations on the concentrations of compounds that can be detected, and upon the spatial resolution that can be achieved. Because of the large number of variables, it is difficult to give anything other than an order-of-magnitude estimate for the concentrations that are required and for the spatial resolution that can be achieved. Typically, however, we can anticipate that, for metabolic studies in vivo, minimum concentrations of 0.2mM and above will be required in order for a metabolite to give a detectable signal.
One of the most remarkable features of magnetic resonance is the extensive range of pulse sequences that have been developed, with a view to enhancing the quality and information content of spectra. For example, innovative pulse sequences have contributed in many ways to improvements in image contrast, spectral localization, suppression of unwanted signals, and visualization of specific structural, biochemical, or functional properties.
The existence of the chemical shift enables us to use NMR to distinguish not only between different molecules, but also between individual atoms within a molecule. When used in conjunction with intensity measurements and spin-spin coupling data, chemical shifts of the spectral lines of a molecules provide a great deal of information about its structure. (Gadian, 2004)
Identify the major observable components in the control samples of human urine (see 1H spectrum obtained for a 'healthy adult' at the session and compare with that of the 7 month old child in the Canavan's disease case study in the lecture notes) - Creatinine (Crn) is already identified for you.
Figure 3. 1H spectra of urine (a) from a 7 month child with Canavan's disease and (b) an age-matched control. (Gadian, 2004)
Canavan's disease is an autosomal recessive disorder in which spongy degeneration of white matter is observed. Several groups have shown a large increase in the NAA/Cr and NAA/Cho ratios in children with Canavan's disease, consistent with enzyme deficiency. The metabolites monitored were those that are present in sufficiently mobile form and at sufficient concentration to give detectable signals.
Figure 3. 1H spectra of urine (a) from a 7 month child with Canavan's disease and (b) an age-matched control. Signals are from creatinine (Crn), hippurate (Hip), betaine (Bet), citrate (Cit), acetate (Ace), lactate (Lac) and alanine (Ala). A large N-acetylaspartate (NAA) is seen in (a) but not in (b).
The urine of patients with Canvan's disease shows an unusual signal that can be attributed to NAA. Quantification of this signal from timed urine samples allows an assessment of the rate at which NAA is being removed from the brain.
Sketch the molecular structures of each of the major components in urine and of Vitamin C. For each molecule indicate which hydrogen atoms are likely to give rise to distinct signals in a water suppressed 1H NMR spectrum (repeat for Vit C and compare with its reference spectrum provided)
Indicates which hydrogen atoms are likely to give rise to distinct signals in a water suppressed 1H NMR spectrum
Components in urine
There are four different types of H but only two signals as two are bound to N
Hippuric acid (Hip)
Lactic acid (Lac)
Oxalic acid (Ox)
Ascorbic Acid (Vitamin C)
(not normally present in urine!)
There are six different types of H but only two signals as four are bound to O
Figure 4. Shows the major spectral changes observed in the spectrum of urine obtained after ingestion of 10g/day Vitamin C over three days.
Identify the major spectral changes observed in the spectrum of urine obtained after ingestion of 10g/day Vitamin C over three days. What information do these spectra provide on the extent of Vitamin C metabolism and on the identities of the major excreted metabolites - this is important - do not gloss over it.
The crn peak stays consistent throughout the 3 day period, as do the other excreted metabolites (Hip, Bet, Cit, Ace). This suggests Vitamin C has no effect on the excretion of other metabolites. The standard and healthy dosage of Vit c is 75 milligrams per day. Therefore at this dosage there is excess Vit c which is unmetabolised and excreted in the urine as shown in fig 4. The diagrams in figure 4 show more Vit c being excreted with each day that passes. Using the standard it is clear to see there is an increase in the peak at the position associated with vitamin C. The area around the peak also generates several smaller peaks. These are not vitamin C but are products with similar structures. These will probably be intermediates in the pathway which breaks down ascorbate acid and contain the same CH2-CH molecular unit intact that was present in the parent ascorbate structure, and this is the bit that gives the NMR fingerprint.
Ascertain (Web of Knowledge or similar search would be appropraite) the generally agreed metabolites (excreted or otherwise) of Vitamin C (there are more than two and this is probably the most important aspect of the report so it needs some investigation!) Discuss whether these could and/or would be identified in the 1H nmr spectrum of urine after a prolonged high dosage of vitamin C. What common feature persists throught the degradative pathway- does this match your results?
The generally agreed metabolites of Vit C are dehydroascorbate (DHAA), 2-O-methyl ascorbate, 2-ketoascorbitol as well as those in figure 5 (L-Threonic acid, Oxalic acid, Lactic acid).
Dehydroascorbate, if not reduced back to ascorbate, decomposes with a half-life of a few minutes, since this
compound is unstable at physiologic pH. The product of the hydrolysis is 2,3-diketo-L-gulonate, which does not possess antiscorbutic effects any more. 2,3-diketo-L-gulonate is decarboxylated to L-xylonate and L-lyxonate. These 5-carbon compounds can enter the pentose phosphate pathway and the L- to D-conversion is suggested to occur through xylitol. Another minor pathway of ascorbate catabolism is a carbon chain cleavage yielding oxalate and 4-carbon intermediates. Pentose phosphate pathway enters the glycolytic/gluconeogenic sequence at triose phosphates and fructose-6-phosphate. Ascorbate and dehydroascorbate, according to the previous assumptions, can be rapidly metabolized to glucose in isolated murine hepatocytes and in HepG2 cells. When glutathione-dependent recycling is inhibited by the oxidant menadione or by the glutathione synthesis inhibitor buthionine sulfoximine, gluconeogenesis from ascorbate is stimulated. The participation of the non-oxidative branch of the pentose phosphate pathway has been demonstrated by the administration of oxythiamine, a thiamine antagonist which inhibits transketolases. In hepatocytes gained from oxythiamine-treated mice glucose production from dehydroascorbate is lower, and a pentose phosphate cycle intermediate, xylulose-5-phosphate is accumulated. This path of ascorbate catabolism could be demonstrated even in cells unable to synthesize ascorbate, i.e., in cells of human origin and in non-hepatic murine cells. In murine and human erythrocytes-which are unable to synthesize glucose (glucose-6-phosphatase is lacking)-ascorbate or dehydroascorbate addition resulted in the increase of lactate, the end product of anaerobic glycolysis. Lactate production could be stimulated by the addition of menadione or inhibited by oxythiamine treatment of the cells indicating that the pentose phosphate pathway is involved in ascorbate catabolism both in hepatocytes and in erythrocytes. These results show that ascorbate does not get lost but is effectively reutilized even in case of diminished recycling and it should be taken into account not only as a vitamin, but also as a source of energy. (Banhegyi, Braun, Csala, Puskas, & Mandl, 1997)
Figure 5. Ascorbate catabolizing pathways in animals. (Banhegyi, Braun, Csala, Puskas, & Mandl, 1997)
It would be hard to identify the metabolites of Vit c in the 1H nmr spectrum of urine after a prolonged high dosage of vitamin C as figure 6 shows a large peak of unmetabolised Vit c which is excreted in the urine. This peak, surrounded by intermediates of the pathway which breaks down ascorbate acid, dominates the 1H nmr spectrum and masks weaker signals. Therefore the metabolites which are produced by the breakdown of some of the Vit c are hard to identify. The common feature which persists throughout the degradative pathway is the CH2-CH molecular unit which is part of all the intermediates within the pathway, and this is the bit that gives the NMR fingerprint. This is shown in figure 6 with several smaller peaks around the Vit C peak. These are the intermediates of the pathway which contain the CH2-CH molecule which is present in the parent ascorbate acid and therefore have a similar structure and appear as peaks around Vit C. These will probably be intermediates in the pathway which breaks down ascorbate acid and contain the same CH2-CH molecular unit intact that was present in the parent ascorbate structure
Comment on the human body's requirement for vitamin C, its role in prevention/treatment of disease (briefly), the required daily intake/doseage, etc. How does this relate to the results dicussed above?
Recommendations for vitamin C intake have been set by various national agencies:
75 milligrams per day: the United Kingdom's Food Standards Agency
The key importance of Vitamin C is supporting the immune system and forming a structural component known as collagen. It is also required for synthesis of the neurotransmitter, required for brain function and mood change. Vitamin C aids in synthesis of a small molecule, carnitine. Carnitine is required for fat transportation to cellular organelles known as mitochondria, potentially, producing energy. Vitamin C has the ability to enhance body's resistance to varied diseases. It aids in stimulating the action of antibodies and immune cells like phagocytes, resulting in a stronger immune system.
Vitamin C metabolite L-threonic acid or its calcium salt, calcium threonate (the form of L-threonic acid found in Ester-C), increases vitamin C uptake of cells. Essentially, with calcium threonate, vitamin C has been shown to be absorbed more quickly, reach higher levels and is excreted more slowly. Now the studies confirm that the vitamin C uptake of the cells is greater with the metabolite L-threonic acid present.
Finally (almost) - identify the advantages and disadvantages of using NMR over other common analytical methods used in Biomedical Sciences (or elsewhere).
In NMR spectroscopy, only a very small excess of the spins are in the low energy state. The net result of this is that NMR is rather insensitive technique relative to many other analytical methods. Typically, even today's spectrometers require a minimum of several nanomoles of material for anaylsis in reasonable times.
Poor sensitivity has been the bane of bioanalytical uses of NMR and increasing NMR sensitivity has been the focus of most of the technical developments that have occurred over the past four decades.
However, in contrast to the low intrinsic sensitivity in the applications of NMR to biofluids, the non-selectivity of NMR makes it a very powerful tool for surveying the molecular content of a sample without prejudging which analytes to search for. This advantage can also be a nuisance. Scarce analytes often need to be measured and although above the limit of detection, these lower level species may be fully or partially obscured by analytes at much higher concentrations. (Gadian, 2004)
A comparison of NMR spectroscopy with HPLC shows a variety of advantages of NMR over HPLC method. The primary advantage of NMR is its efficiency due to the lack of any preparation times. The analyte has to be weighed and dissolved in the solvent only and afterwards the analyte can be measured immediately. The experimental time depends on the concentration of the analyte. Using HPLC for the determination of an analyte much time has to be spent for the equilibration of the column. The column has to be washed every day after the measurements have been taken to prolong the lifetime of the column. When using the HPLC technique, often much time has to be spent for sample preparation e.g. derivatization of the analyte. A further disadvantage is the large amount of solvent necessary for the HPLC separation. NMR is also more efficient than the conventional HPLC techniques. (Wawer, Holzgrabe, & Diehl, 2008)