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1H NMR spectroscopic survey of plasma and erythrocytes from selected marsupials and domestic animals of Australia
Abstract
1.1. 1H NMR spectra were acquired from whole plasma, intact erythrocytes, and ultrafiltrates of erythrocytes from nine native and eight introduced (domestic) Australian animals; single-pulse, spin-echo and 2-dimensional spectra were obtained. The aim was to detect and at least semi-quantify metabolites in the samples and compare the profiles amongst the species.2.2. The Australian natives that were studied were all marsupials: greater brown bandicoot; bettong; eastern grey kangaroo; red kangaroo; koala; possum; red necked pademelon; Tammar wallaby; and wombat. The introduced mammals that were studied were: cat; cattle; dog; goat; horse; pig; rabbit; and sheep.3.3. Because of the range of habitats and diets amongst the animals, it was postulated that the concentrations of the common metabolites in the blood would show marked differences and that there would also be some metabolites that were peculiar to a given animal. There were several major differences in the spectra: in the spectra of plasma, the glycoprotein and lipoprotein resonances showed the largest inter-species variation, whereas the most dramatic finding from the spectra of erythrocytes was a very high concentration of lysine in the cells from the Tammar wallaby.
... Though relatively few, these reports have provided valuable information in comparative biochemistry; e.g. almost negligible amounts of ATP are found in the erythrocytes of echidna and platypus (Ornithorynchus anatinus; Kim et al. 1981;Isaacks et al. 1984), high concentrations of 2,3-bisphosphoglycerate (2,3-DPG) in the erythrocytes of wombat (Vombatus ursinus; Agar et al. 1989) and high concentrations of lysine in those of the Tammar wallaby (Macropus eugenii; Agar et al. 1991). ...
... We recently presented a 1H NMR spectroscopic survey of plasma and red blood cells (RBC) from selected domestic and native animals of Australia (Agar et al. 1991). In continuing this work we examined four more species; echidna (Tachyglossus aculeatus), Tasmanian devil (Sarcophilus harrisii), tree kangaroo (Dendrolagus lumholtzi) and a native aquatic bird, the 'little' penguin (Eudyptula minor). ...
... The methods used for preparing haemolysates and ultrafiltrates were as described previously (Agar et al. 1991). Perchloric acid (PCA) extracts were prepared according to the method of Beutler (1984). ...
1H and 31P NMR spectra were acquired from the whole plasma and ultrafiltrates of erythrocytes (RBC) from eight Australian native animals. Various metabolites contained in the samples were identified from their NMR spectra and their concentrations were determined where possible. The key observations were as follows. (1) High lysine concentrations in the RBC from Tammar wallaby (8 mmol/(L RBC)) were confirmed. (2) A resonance at 3.17 in the 1H spin-echo NMR spectrum of koala RBC was reassigned to the N+-(CH3)3 of betaine and not to ergothioneine as previously claimed. (3) A high concentration (10 mmol/ (L RBC)) of taurine was observed in the RBC from little penguin. (4) The lack of ATP in the RBC of echidna, reported previously on the basis of standard biochemical assays, was confirmed directly by NMR spectroscopy. (5) The highest ergothioneine concentrations in the RBC of the eight species of animal were in the wombat (1.76 mmol/(L RBC)).
... Since authentic samples are not always available and, if added for verification, inevitably compromise part of the sample under investigation, the latter approach is clearly preferable. While there have been numerous studies involving monitoring of specific processes by heteronuclear NMR (e.g., 15), sensitivity considerations have dictated that most literature determinations of the structures of components of biofluids have been based on 1 H NMR data (16), with 13 C data only used as supplementary information in some studies (14), for the characterization of specific classes of compounds in extracts (17,18), or to circumvent limited dispersion of 1 H chemical shifts when samples have been available in sufficiently large quantities (19). ...
... Chemical shifts for all amino acid resonances are consistent with 1 H (23) and 13 C (24) literature data, as are ( 1 H, 13 C) shifts for lactate and acetate (16,24), ethanol, acetaldehyde and succinate (25,24), ␥-aminobutyric acid (GABA) (26,24) and glycine betaine (betaine) (16,27). The 1D 31 P NMR spectrum was dominated by a strong resonance with no 1 H correlations, assumed to be inorganic phosphate; minor resonances were just distinguishable from the baseline. ...
... Chemical shifts for all amino acid resonances are consistent with 1 H (23) and 13 C (24) literature data, as are ( 1 H, 13 C) shifts for lactate and acetate (16,24), ethanol, acetaldehyde and succinate (25,24), ␥-aminobutyric acid (GABA) (26,24) and glycine betaine (betaine) (16,27). The 1D 31 P NMR spectrum was dominated by a strong resonance with no 1 H correlations, assumed to be inorganic phosphate; minor resonances were just distinguishable from the baseline. ...
The yeast, Cryptococcus neoformans var. neoformans is a major contributor to the morbidity and mortality experienced by the immunosuppressed population. With a view to providing better treatment, identification of cryptococcal virulence factors is an important goal, with most effort to date directed toward the significance of structural variations in the polysaccharide capsule. The present work describes the characterization of supernatants obtained from cryptococcal cultures. This was achieved by thorough identification of the spin systems of individual metabolites through both homonuclear and heteronuclear NMR experiments that circumvented the difficulties imposed by limited dispersion and a range of concentrations in different cultures covering more than 3 orders of magnitude. More than 30 metabolites, including amino acids, alditols, nucleosides, acetate, and ethanol, were identified by their (1)H and (13)C chemical shifts and observed long-range correlations. The possible contribution of some detected substances to the pathogenicity of Cryptococcus neoformans is discussed. Magn Reson Med 42:442-453, 1999.
... Cartilage consists of a complex network of dif ferent polysaccharides and proteins (mainly colla gen II), which are not extractable from the tissue by buffer. Due to the metabolic activity of carti lage cells (chondrocytes), some physiologically re levant molecules are also detectable in cartilage supernatants (Agar et al., 1991). Fig. 2 shows typi cal spin-echo (a) and pulse-acquired (b) 'H-NM R (ppm) Fig. 2. 'H-NMR spectra of pig articular cartilage (0.5 g wet weight) after incubation in 2 ml 50 mM phosphate buffer containing 154 mM NaCl at 37 °C for two hours, (a) was recorded using the Hahn spin-echo sequence with a delay t=60 ms resulting in a turning of phases of coupled spin-systems, whereas (b) was obtained under analogous conditions as single pulse spectrum (cut off for clarity due to its high intensity). ...
... In supernatants of cartilage samples treated with collagenase (3b) an intense singlet at 3.55 ppm is observed after papain treatment. This resonance obviously results from glycine (Agar et al., 1991), which is present in constant, high pro portions (about 33%) in all cartilage collagens. The glycine singlet is easily detectable by NM R (it contains only a single kind of protons) and pro vides a reliable indicator of collagen degradation. ...
Rheumatic diseases are accompanied by a progradient diminution of the cartilage layer. Unfortunately, degradation mechanisms (role of different enzymes and reactive oxygen species) are not yet understood. Since nuclear magnetic resonance (NMR) spectroscopy was often used for the investigation of synovial fluids, the aim of the present work was to detect cartilage degradation products upon proteolytic digest of cartilage. Cartilage samples were incubated at 37 degrees C with phosphate buffer in the absence or presence of different proteases (collagenase, trypsin and papain). Supernatants were subsequently assayed towards their content of carbohydrate and protein degradation products by NMR (1H- and 13C-) and MALDI-TOF mass spectrometry. Intense resonances of relatively mobile N-acetyl side chains (ca. 2.01 ppm) of polysaccharides of cartilage were only detectable on digestion with papain. The appearance of these resonances indicates intense degradation of the core protein of proteoglycan aggregate of cartilage, whereby polysaccharides are released. Additionally, broad resonances at 0.85 ppm arising from collagen degradation products were observed upon the action of all applied proteases. However, glycine as a marker of exhaustive collagen degradation was only observed, if cartilage was digested by collagenase. Using more vigorous incubation conditions, additionally high-abundant amino acids of collagen (proline, hydroxyproline) could be detected in the 13C-NMR- and the MALDI spectra. The observed differences are correlated with the different selectivities of the applied enzymes. It is concluded that NMR allows the detection of characteristic protein and polysaccharide degradation products. The observed differences may be of some relevance for the diagnosis of rheumatic diseases.
... Identification of metabolites. The preliminary identity of the peaks used in PCDA was determined with an internal specialized database of 400 compounds for compound identification in rat urine, an external database (http://www.liu.se/hu/mdl/main/) ( Agar et al., 1991;Fan, 1996a,b;Govindaraju et al., 2000) and several literature comparisons ( Bollard et al., 2005;Clayton et al., 2006;Robertson et al., 2000). These assignments are not definitive and should be handled with care because small chemical shifts due to pH differences between the spectra in the internal database and the spectra in our study can be present. ...
... It reacted weakly with ninhydrin, consistent with a mono-methylamine (this and amino acids react with ninhydrin, while di-and trimethylamines do not). In the NMR, this solute had a large singlet peak at 2.73 ppm, in the range of N-methyl hydrogens on monomethylamines (e.g., N-methyl hydrogens of sarcosine and methyltaurine are at 2.71 and 2.79 ppm, respectively) (Agar et al., 1991;. Another singlet peak was at 3.37 ppm and was unidentifiable. ...
Hydrostatic pressure can inhibit protein functions. Many deep-sea proteins have evolved resistance to this, but not all. Recently we have found unusual osmolyte compositions in deep-sea fishes and other animals. The protein-stabilizer trimethylamine N-oxide (TMAO), under 80 mmol/kg wet wt in most shallow teleosts, increases with depth in white muscles of several families of teleosts (up to 288 mmol/kg at 2900 m). Similarly, deep-sea skates have more TMAO (and less urea) than do shallow relatives. Here we report that red muscle of deep-sea macrourids contains 93-118 mmol/kg wet wt TMAO (less than white muscle, but more than shallow species) plus large amounts of a possible (but unidentified) methylamine. We hypothesized that methylamines counteract effects of pressure on proteins, and have tested TMAO and pressure with lactate dehydrogenase, pyruvate kinase, and actin, all pressure-sensitive proteins. 200- 250 mM TMAO (but not glycine) protected ligand binding, protein stability, and polymerization against pressure inhibition. Here, to test TMAO in living cells, we grew yeast under pressure. After 1 hr at 71 MPa, 3.5 hr at 71 MPa, and 17 hr at 30 MPa, 150 mM TMAO (but not glycine) generally doubled the number of cells that formed colonies. These results support the hypothesis.
... Peaks in the 'H NMR spectra were identified by comparison (Table 1) with literature values of the chemical shifts (after correction of TSP shifts to DSS values) and coupling pattern (e.g., 10,[18][19][20][21], with the NMR spectra of authentic standard compounds, and by adding a small amount of authentic compound (22) to the sample, followed by the acquisition of an additional spectrum. ...
Urinary N,N,N-trimethylglycine (betaine) and N,N-dimethylglycine (DMG) have been identified and quantified for clinical purposes by proton nuclear magnetic resonance (1H NMR) measurement in previous studies. We have assessed these procedures by using both one-dimensional (1-D) and 2-D NMR spectroscopy, together with pH titration of urinary extracts to help assign 1H NMR spectral peaks. The betaine calibration curve linearity was excellent (r = 0.997, P = 0.0001) over the concentration range 0.2-1.2 mmol/L, and CVs for replicate betaine analyses ranged from 7% (n = 10) at the lowest concentration to 1% (n = 9) at the highest. The detection limit for betaine was < 15 mumol/L. Urinary DMG concentrations were substantially lower than those of betaine. Urinary betaine and DMG concentrations measured by 1H NMR spectroscopy from 13 patients with premature vascular disease and 17 normal controls provided clinically pertinent data. We conclude that 1H NMR provides unique advantages as a research tool for determination of urinary betaine and DMG concentrations.
... Fig. 1 shows a typical 'H-NMR spin-echo spectrum (Fig. 1 a) and the corresponding single-pulse spectrum ( Fig. 1 b) of the high-field region from pig articular cartilage incubated with sodium phosphate buffer prepared in D,O for 1 h at 37°C. Most resonances appearing in the spectra arise from well known, low-molecular-mass chemical substances (Agar et al., 1991). The use of 3-(trimethylsilyl)-propane-1-sulfonate as a water-soluble internal reference allowed the determination of their concentrations. ...
The action of sodium hypochlorite on pig articular cartilage was studied by 1H-NMR spectroscopy to model some aspects of degradation processes of cartilage during rheumatoid arthritis. Two effects of NaOCl on cartilage polysaccharides have been observed. Hypochlorous acid causes an enhanced release of oligomeric polysaccharides from cartilage. The second effect concerns the degradation of N-acetyl side chains of carbohydrates to acetate via a chlorinated transient product.
Signal intensities for N-acetyl groups (≈2.0 ppm) increase during the first 2 h of incubation of cartilage with NaOCl. Then they decrease again. However, acetate (1.90 ppm) as the final product of degradation of N-acetyl side chains increases continuously over the period of incubation with NaOCl.
In addition to polysaccharides, effects of NaOCl were only observed in cartilage samples on amino acids like alanine. The alanine resonance disappeared already at NaOCl concentrations where only small effects on cartilage polysaccharides have been observed.
... Inherited high K + red cells in the dog show high accumulation of glutamate, aspartate and glutamine, due to a Na + -concentration gradient induced by Na + K + -ATPase in the membrane [5,9]. On the other hand, in marsupials only one report on an NMR study of RBC and plasma is available which provides information on amino acid concentrations and metabolism of this group [1]. The NMR study revealed a high lysine concentration in the RBC of Tammar Wallaby. ...
It was recently coincidentally discovered, using 1H NMR spectroscopy, that the erythrocytes of two species of Australian marsupials, Tammar Wallaby (Macropus eugenii) and Bettong (Bettongia penicillata), contain relatively high concentrations of the essential amino acid lysine (Agar NS, Rae CD, Chapman BE, Kuchel PW. Comp Biochem Physiol 1991;99B:575-97). Hence, in the present work the rates of transport of lysine into the erythrocytes from the Common Brushtail Possum (Dactylopsilia trivirgata) and Eastern Grey Kangaroo (Macropus giganteus) (which both have low lysine concentrations), and Tammar Wallaby were studied, to explore the mechanistic basis of this finding. The concentration-dependence of the uptake was studied with lysine alone and in the presence of arginine, which may be a competitor of the transport in some species. In relation to GSH metabolism, glutamate uptake was determined in the presence and absence of Na+. The data was analysed to yield estimates of the maximal velocity (Vmax) and the Km in each of the species. Erythrocytes from Tammar Wallaby lacked saturable lysine transport in contrast to the other two species. The glutamate uptake was normal in all three animals for adequate GSH biosynthesis.
... The only phosphorylated compound with sufficient signal for measuring D at all diffusion times was arginine phosphate, although we were able to reliably measure ATP diffusion in some cases (see below). Peak assignments in 1 H-spectra were made by spiking muscle extracts with putative compounds and by comparison of our spectra to the chemical shift data given in Agar et al. (1991). The betaine peak at 3.2 p.p.m. was large and well resolved in 1 H-NMR spectra, and D could be measured at all diffusion times for this metabolite (Fig. 2). ...
The time- and orientation-dependence of metabolite diffusion in giant muscle fibers of the lobster Panulirus argus was examined using (31)P- and (1)H-pulsed-field gradient nuclear magnetic resonance. The (31)P resonance for arginine phosphate and the (1)H resonances for betaine, arginine/arginine phosphate and -CH(2)/-CH groups were suitable for measurement of the apparent diffusion coefficient, D. Diffusion was measured axially, D(//), and radially, D( perpendicular ), in fibers over diffusion times of 20 to 300 ms. Diffusion was strongly anisotropic, and D(//) was higher than D( perpendicular ) at all times. Radial diffusion decreased with time until a steady-state value was reached at a diffusion time of approximately 100 ms. Changes in D( perpendicular ) occurred over a time scale that was consistent with previous measurements from fish and mammalian muscle, indicating that diffusion is hindered by the same types of barriers in these diverse muscle types. The time dependence indicated that the sarcoplasmic reticulum is the principal intracellular structure that inhibits mobility in an orientation-dependent manner in skeletal muscle. The abdominal muscles in P. argus are used for anaerobic, burst contractions during an escape maneuver. The fact that these muscle fibers have diameters that may exceed hundreds of microns in diameter, and nearly all of the mitochondria are localized near the sarcolemmal membrane, suggests that barriers that hinder radial diffusion of ATP equivalents may ultimately limit the rate of post-contractile recovery.
... Identification of metabolites. The preliminary identity of the peaks used in PCDA was determined with an internal specialized database of 400 compounds for compound identification in rat urine, an external database (http://www.liu.se/hu/mdl/main/) ( Agar et al., 1991;Fan, 1996a,b;Govindaraju et al., 2000) and several literature comparisons (Bollard et al., 2005;Clayton et al., 2006;Robertson et al., 2000). These assignments are not definitive and should be handled with care because small chemical shifts due to pH differences between the spectra in the internal database and the spectra in our study can be present. ...
(1)H nuclear magnetic resonance (NMR) spectroscopy of rat urine in combination with pattern recognition analysis was evaluated for early noninvasive detection of toxicity of investigational chemical entities. Bromobenzene (B) and paracetamol (P) were administered at five single oral dosages between 2 and 500 mg/kg and between 6 and 1800 mg/kg, respectively. The sensitivity of the proposed method to detect changes in the NMR spectra 24 and 48 h after single dosing was compared with histopathology and biochemical parameters in plasma and urine. Both B and P applied at the highest dosages induced liver necrosis and markedly increased aspartate aminotransferase (AST) and alanine aminotransferase (ALT) plasma levels. At dosages of 125 mg/kg B and 450 mg/kg P, liver necrosis and changes in AST and ALT were less pronounced, while at lower dose levels these effects could not be detected. Changes in kidney pathology or standard urine biochemistry were not observed at any of these dosages. Evaluation of the total NMR dataset showed 80 signals to be sensitive for B and P dosing. Principal component analysis on the reduced dataset revealed that NMR spectra were significantly different at dosages above 8 mg/kg (B) and 110 mg/kg (P) at both sampling times. This implies a 4- to 16-fold increased sensitivity of NMR versus histopathology and clinical chemistry in recognizing early events of liver toxicity.
Moxidectin (MOX) is a macrocyclic lactone used to eliminate endo and ectoparasites in many mammalian species. It is notably the active ingredient of the anti-parasitic drug Cydectin®, manufactured by Virbac, and is frequently used to treat sarcoptic mange in Australian wildlife. Protein binding plays a significant role in the efficacy of a drug, as the unbound/free drug in plasma ultimately reflects the pharmacologically relevant concentration. This study aimed to investigate the free drug percentage of Moxidectin after in vitro spiking into the sera of four sarcoptic mange-susceptible Australian wildlife species; the koala (Phascolarctos cinereus), the bare-nosed wombat (Vombatus ursinus), the eastern grey kangaroo (Macropus giganteus), and the mountain brushtail possum (Trichosurus cunninghami). Three concentration points of MOX were tested for each individual: 20pg/µL, 100pg/µL and 500pg/µL. Serum from five individuals of each species underwent an equilibrium dialysis followed by liquid chromatography tandem mass spectrometry (LC-MS/MS). The results showed an atypical concentration dependent binding across all species, where free drug percentage decreased as MOX concentration increased. In addition, wombats showed significantly lower free drug levels. These findings call for further research into the mechanisms of moxidectin protein binding to help understand MOX pharmacokinetics in marsupials.
Coenzymes of cellular redox reactions and cellular energy mediate biochemical reactions fundamental to the functioning of all living cells. Despite their immense interest, no simple method exists to gain insights into their cellular concentrations in a single step. We show that a simple 1H NMR experiment can simultaneously measure oxidized and reduced forms of nicotinamide adenine dinucleotide (NAD+ and NADH), oxidized and reduced forms of nicotinamide adenine dinucleotide phosphate (NADP+ and NADPH), adenosine triphosphate (ATP) and its precursors, adenosine diphosphate (ADP) and adenosine monophosphate (AMP), using mouse heart, kidney, brain, liver and skeletal muscle tissue extracts as examples. Combining 1D/2D NMR experiments, chemical shift libraries, and authentic compound data, reliable peak identities for these coenzymes have been established. To assess this methodology, cardiac NADH and NAD+ ratios/pool sizes were measured using mouse models with a cardiac specific knockout of the mitochondrial Complex I Ndufs4 gene (cKO) and cardiac-specific overexpression of nicotinamide phosphoribosyltransferase (cNAMPT) as examples. Sensitivity of NAD+ and NADH to cKO or cNAMPT was observed, as anticipated. Time dependent investigations showed that the levels of NADH and NADPH diminish by up to ~50% within 24 h; concomitantly, NAD+ and NADP+ increase proportionately; however, degassing the sample and flushing the sample tubes with helium gas halted such changes. The analysis protocol along with the annotated characteristic fingerprints for each coenzyme is provided for easy identification and absolute quantification using a single internal reference for routine use. The ability to visualize the ubiquitous coenzymes fundamental to cellular functions, simultaneously and reliably, offers a new avenue to interrogate the mechanistic details of cellular function in health and disease.
Using the method of nuclear magnetic-resonance spectroscopy, lactic acid, alanine, and aspartate were revealed in the coelomic fluid of the black sea urchin Mesocentrotus nudus, which indicates anaerobic metabolism in echinoderms. Energy metabolism of this type is intensified in stressful situations, when the inflow of oxygen into cells becomes insufficient. This compensatory response of energy metabolism is an important mechanism for adaptation to changing environmental conditions.
Rheumatic diseases are accompanied by a progressive destruction of the cartilage layer of the joints. Despite the frequency of the disease, degradation mechanisms are not yet understood and methods for early diagnosis are not available. Although some information on pathogenesis could be obtained from the analysis of degradation products of cartilage supernatants, the most direct information on degradation processes would come from the native cartilage as such. We have used 1H as well as 13C HR-MAS (high resolution magic angle spinning) NMR spectroscopy to obtain suitable line-widths of NMR resonances of native cartilage. 1D and 2D NMR spectra of native cartilage were compared with those of enzymatically-treated (collagenase and papain) samples. In the 1H NMR spectra of native cartilage, resonances of polysaccharides, lipids and a few amino acids of collagen were detectable, whereas the 13C NMR spectra primarily indicated the presence of chondroitin sulfate. Treatment with papain resulted only in small changes in the 1H NMR spectrum, whereas a clear diminution of all resonances was detectable in the 13C NMR spectra. On the other hand, treatment with collagenase caused the formation of peptides with an amino acid composition typical for collagen (glycine, proline, hydroxyproline and lysine). It is concluded that the HR-MAS NMR spectra of cartilage may be of significance for the investigation of cartilage degradation since they allow the fast evaluation of cartilage composition and only very small amounts of sample are required.
Methylamines are frequently present in high concentrations in biological samples, but their separation and quantification
are difficult. Data presented show that methylamines commonly occurring in biological material can be uniquely identified
and quantified by proton nuclear magnetic resonance spectroscopy by recording spectra at both neutral and acid pH. Use of
a high sensitivity probe permits this analysis even in the presence of high water concentrations, allowing accurate quantification
with minimum preparative technique. The method was tested on tissues of the dogfish. Trimethylamine oxide was found in amounts
ranging from 42 mmol kg−1 fresh weight in liver, up to 115 mmol kg−1 fresh weight in heart. Betaine was found to range from 10 mmol kg−1 fresh weight in liver to 49 mmol kg−1 fresh weight in brain. Creatine was not found in heart or liver, but was present in body wall muscle and in brain. Further
analysis using high-performance liquid chromatography allowed determination of urea/methylamine ratios, which ranged from
1.9 in liver to 3.7 in body wall muscle.
Rheumatic diseases are accompanied by a progressive destruction of the cartilage layer of the joints. Despite the frequency
of the disease, degradation mechanisms are not yet understood and methods for early diagnosis are not available. Although
some information on pathogenesis could be obtained from the analysis of degradation products of cartilage supernatants, the
most direct information on degradation processes would come from the native cartilage as such. We have used1H as well as13C HR-MAS (high resolution magic angle spinning) NMR spectroscopy to obtain suitable line-widths of NMR resonances of native
cartilage. ID and 2D NMR spectra of native cartilage were compared with those of enzymatically-treated (collagenase and pa
pain) samples. In the1H NMR spectra of native cartilage, resonances of polysaccharides, lipids and a few amino acids of collagen were detectable,
whereas the13C NMR spectra primarily indicated the presence of chondroitin sulfate. Treatment with papain resulted only in small changes
in the1H NMR spectrum, whereas a clear diminution of all resonances was detectable in the13C NMR spectra. On the other hand, treatment with collagenase caused the formation of peptides with an amino acid composition
typical for collagen (glycine, proline, hydroxyproline and lysine). It is concluded that the HR-MAS NMR spectra of cartilage
may be of significance for the investigation of cartilage degradation since they allow the fast evaluation of cartilage composition
and only very small amounts of sample are required. © 2001 Elsevier Science B.V. All rights reserved.
Although extensive studies have been performed on human erythrocytes, there is a shortage of information on marsupial erythrocytes.
Studies on haematology and biochemistry are useful in the ecomanagement of these animals especially those in wildlife parks
and zoos. The present review summarises our findings from ∼30 species of marsupials. As marsupials show great diversity in
physical and behavioural characteristics, it is not surprising that examination of their red blood cells reveals variation
in the biochemical features. Many variations in red cell biochemistry appear to be species specific and, although interesting,
are too numerous to list here and have been discussed in the relevant sections. Red blood cells of several species of marsupial
differ from those in placental mammals by having higher haemoglobin concentration and haematocrit, lower ATP and higher 2,3-diphosphoglycerate
concentrations. These features are consistent across the majority of marsupial species, but the relevance of these variations
from red cell metabolism in the placental mammals is unknown.
The phospholipid classes of erythrocyte membranes and plasma from several domestic animals and marsupials were quantified by 31P NMR using detergents. Washed erythrocyte samples were thoroughly haemolysed by tip-sonication and dissolved in sodium cholate; plasma samples were dissolved in Triton X-100. The species studied were: common wombat (Vombatus ursinus), black-striped wallaby (Macropus dorsalis), bandicoot (Isoodon macrocarpus), Eastern grey kangaroo (Macropus giganteus), Tammar wallaby (Macropus eugenii), sheep (Ovis aries), goat (Capra hircus), cattle (Bos taurus), horse (Equus caballus), dog (Canus familiaris) and rabbit (Orytolagus caniculus). There were considerable species variations in the relative abundance of erythrocyte and plasma phospholipid classes. The variations may be attributed to the habitats and diets of the animals as well as to their phylogenetic differences.
Although it has been recently shown by the use of1H nuclear magnetic resonance (1H NMR) spectroscopy that enzymatically formed hypochlorous acid (HOCl/Cl⊖) reacts with carbohydrates containingN-acetyl groups under the formation of acetate and presumably chloramines, mechanistical aspects of this reaction remain unknown. Since NMR spectroscopy suffers a lot from low sensitivity especially toward polymeric components giving less marked resonances, we used here infrared (IR) spectroscopy to characterize the products of the reaction between hypochlorite and chondroitin sulfate. NMR is used for means of comparison. The reaction of HOCl/Cl⊖with chondroitin sulfate does not change most regions of the IR spectrum of the polysaccharide markedly. Whereas most vibrational bands remain nearly unchanged toward their position and their relative intensities, an additional band at 975 cm−1is clearly detectable. This band is in good accordance with the proposed formation of a N-Cl-bond in chondroitin sulfate upon HOCl treatment. Additionally, NMR experiments clearly indicate that chloramine formation is accompanied by the formation of acetate. For comparison, taurine (2-amino-1-ethanesulfonic acid) was also investigated toward its reactivity with HOCl/Cl⊖. An analogous band at 975 cm−1was found which unequivocally indicates the formation of a chloramine. NMR also detects changes upon this reaction, but these changes cannot be exactly assigned to chloramine formation. Thus, we conclude that IR spectroscopy is most suitable for the detection of chloramines due to its relatively high sensitivity and the possibility to detect directly N-Cl-groups.
Pulsed field-gradient spin-echo (PGSE) NMR spectroscopy via q-space plots can characterize erythrocyte shapes and their evolution. The present study employed PGSE NMR to investigate shape reversion from advanced echinocytic to normal discocytic shapes due to depletion and then readdition of Mg(2+). In q-space plots of the data, the diffusion-diffraction minima disappeared for Mg(2+)-depleted erythrocytes and reappeared during the shape recovery process, but with lower definition than for control cells. Shape estimates from PGSE NMR spectra and light microscopy were in excellent agreement after application of a scaling/correction factor. (31)P NMR was used to probe the biochemical processes activated in erythrocytes after depletion or addition of Mg(2+); it showed the activation of the nonoxidative part of the pentose phosphate pathway. Experimental conditions were optimized to bypass this pathway without any influence on the q-space plots. The release of choline from phosphatidylcholine in the outer leaflet of the plasma membrane of the cells, observed using (1)H spin-echo NMR, showed a higher rate for shape-recovered than for control cells. This points to a change in phospholipid asymmetry in the plasma membrane. This variation in asymmetry affected the mean cell shape and hence influenced the average alignment of the erythrocytes with the static magnetic field and so affected the shapes of the q-space plots.
NMR spectroscopy was used to identify and quantify compounds in extracts prepared from mature trophozoite-stage Plasmodium falciparum parasites isolated by saponin-permeabilisation of the host erythrocyte. One-dimensional (1)H NMR spectroscopy and four two-dimensional NMR techniques were used to identify more than 50 metabolites. The intracellular concentrations of over 40 metabolites were estimated from the (1)H NMR spectra of extracts prepared by four extraction methods: perchloric acid, methanol/water, methanol/chloroform/water, and methanol alone. The metabolites quantified included: the majority of the biological alpha-amino acids; 4-aminobutyric acid; mono-, di- and tri-carboxylic acids; nucleotides; polyamines; myo-inositol; and phosphocholine and phosphoethanolamine. The parasites also contained a significant concentration (up to 12 mM) of the exogenous buffering agent, HEPES. Although the metabolite profiles obtained with each extraction method were broadly similar, perchloric acid was found to have significant advantages over the other extraction media.
Recently, it has been reported that hypochlorous acid (HOCl), a special product of neutrophil myeloperoxidase, degrades N-acetyl groups of N-acetylglucosamine, chondroitin sulfate, hyaluronic acid, and minced articular cartilage via a transient product to acetate. This work concerns 1H NMR investigations of synovial fluids of patients with rheumatoid arthritis (RA). Synovial fluids of patients with severe forms of this disease are characterized by enhanced 1H NMR signals for N-acetyl groups (approximately 2.0 ppm) and acetate (1.90 ppm) and the appearance of a broad but less intense signal at 2.35 ppm. It is likely that this signal corresponds to the transient, chlorinated product of degradation of N-acetyl groups by hypochlorous acid. Moreover, 1H NMR signal intensities of N-acetyl groups and acetate strongly correlate with the myeloperoxidase activities in synovial fluids from patients with rheumatoid arthritis. These results have been confirmed by treatment of native sheep synovial fluid with sodium hypochlorite, resulting in the formation of the same resonances as observed in pathologically changed synovial fluids from humans. Thus, it is concluded that HOCl plays an important role for the cartilage degradation during rheumatoid arthritis.
We recently identified phospholipase activity as a potential virulence factor of Cryptococcus neoformans. We have now defined the nature of the phospholipase activity produced by a clinical isolate of C. neoformans var. neoformans, under native conditions, by 1H and 31P nuclear magnetic resonance (NMR) spectroscopy and thin-layer chromatography (TLC) of radiolabelled substrates. Glycerophosphocholine was identified by NMR spectroscopy as the sole phospholipid degradation product of the reaction between substrate phosphatidylcholine (PC) and cryptococcal culture supernatants indicating the presence of phospholipase B (PLB). No lysophosphatidylcholine (lyso-PC) or products indicative of phospholipase C, phospholipase D, or other lipase activity were identified. Use of PC and lyso-PC containing radiolabelled acyl chains and separation of products by TLC confirmed the PLB and lysophospholipase (LPL) activities. Lysophospholipase transacylase (LPTA) activity was identified by the formation of radioactive PC from lyso-PC. Extracellular enzyme production was maximal after 6 to 10 h in fresh medium. Assay conditions were optimized for pH, linearity with time, enzyme concentration, and saturation by substrates to allow comparison with phospholipases from other organisms. LPL activity was 10- to 20-fold greater than PLB activity, with mean (+/- standard deviation) specific activities of 34.9 +/- 7.9 and 3.18 +/- 0.2 micromol of substrate hydrolyzed per min per mg of protein, respectively. The response of PLB to increasing substrate concentrations was bimodal, whereas inhibition of LPL and LPTA activities occurred at concentrations of substrate lyso-PC greater than 200 microM. Enzyme activities were stable at acid pH (3.8), with pH optima of 3.5 to 4.5. Activities were unchanged in the presence of exogenous serine protease inhibitors, divalent cations, and EDTA. We conclude that C. neoformans produces highly active extracellular PLB, LPL, and LPTA under native conditions.
The action of sodium hypochlorite (NaOCl) on bovine nasal cartilage was studied by proton nuclear magnetic resonance ( ¹ H-NMR) spectroscopy in order to model degradation processes of cartilage caused by neutrophil-derived hypochlorous acid.
Nasal cartilage was chosen as a mean of comparison because it differs from articular cartilage in its composition. It contains some more proteoglycans, i.e. polymeric carbohydrates and less collagen than articular cartilage. This is important for studying the influence of hypochlorous acid on cartilage components (collagen and polysaccharides). Cartilage samples were incubated at 37 °C with phosphate buffer in the presence or absence of NaOCl. Supernatants were collected and assayed by NMR-spectroscopy. In the presence of pure phosphate buffer, the supernatants of bovine nasal cartilage were less rich in low molecular mass metabolites (e.g. amino acids, lactate) than articular cartilage. However, intense signals for highly mobile N acetyl groups of cartilage polysaccharides were detectable in nasal cartilage.
NaOCl caused an increase in signals for acetate and formiate. Signals for N-acetyl groups rose only during the first 25 minutes of incubation with NaOCl. Then, their concentration decreased markedly. These changes were related to an enhanced release of Chondroitinsulfate from nasal cartilage
It was investigated to what extent isolated, monomeric and polymeric carbohydrates as well as cartilage specimens are affected by hydroxyl radicals generated by gamma-irradiation or Fenton reaction and what products can be detected by means of NMR spectroscopy. Resonances of all protons in glucose and other monosaccharides as well as carbon resonances in 13C-enriched glucose were continuously diminished upon gamma-irradiation. Formate and malondialdehyde were found as NMR detectable products in irradiated glucose solutions under physiologically relevant (aerated) conditions. In polysaccharide solutions (e.g. hyaluronic acid) gamma-irradiation and also treatment with the Fenton reagent caused first an enhancement of resonances according to mobile N-acetyl groups at 2.02 ppm. This indicates a breakdown of glycosidic bonds in polysaccharides. Using higher radiation doses or higher concentrations of the Fenton reagent formate was also detected. The same sequence of events was observed upon treatment of bovine nasal cartilage with the Fenton reagent. First, glycosidic linkages in cartilage polysaccharides were cleaved and subsequently formate was formed. In contrast, collagen of cartilage was affected only to a very low extent. Thus, HO-radicals caused the same action on cartilage as on isolated polymer solutions, inducing a fragmentation of polysaccharides and the formation of formate.
Pulsed field gradient (PFG) nuclear magnetic resonance (NMR) was used to investigate the self-diffusion behavior of water molecules in cartilage, polymeric cartilage components, and different model polymers. The short-time self-diffusion coefficients (diffusion time delta approximately/= 13 msec) are found to decrease steadily with decreasing water content. This holds equally well for cartilage and cartilage components. The short-time diffusion coefficients are subjected to a rather nonspecific obstruction effect and mainly depend on the water content of the sample. The long-time diffusion coefficients in cartilage (delta approximately/= 500 msec), however, reflect structural properties of this tissue. Measurements with varying observation times as well as experiments involving enzymatic treatment of articular cartilage suggest that the collagenous network in cartilage is likely to be responsible for the observed restricted diffusion.
Although neutrophilic granulocytes clearly contribute to cartilage degradation in rheumatic diseases, it is unclear if reactive oxygen species (ROS) or proteolytic enzymes are the most important components in cartilage degradation and how they interact.
Neutrophils were stimulated by chemicals conferring a different degree of ROS formation and enzyme release. Supernatants of neutrophils were incubated with thin slices of pig articular cartilage. Supernatants of cartilage were assayed by NMR spectroscopy, MALDI-TOF mass spectrometry and relevant biochemical methods. Stimulation conditions of neutrophils correlated well with the extent of cartilage degradation. Due to the release of different enzymes, cartilage degradation could be best monitored by NMR since mainly low-mass degradation products were formed. Astonishingly, the suppression of the formation of ROS resulted in decreased cartilage degradation.
ROS formed by neutrophils are not directly involved in cartilage degradation but influence the activity of proteolytic enzymes, which are the main effectors of cartilage degradation.
Organic osmolytes, solutes that regulate cell volume, occur at high levels in marine invertebrates. These are mostly free amino acids such as taurine, which are "compatible" with cell macromolecules, and methylamines such as trimethylamine oxide, which may have a nonosmotic role as a protein stabilizer, and which is higher in many deep-sea animals. To better understand nonosmotic roles of osmolytes, we used high-performance liquid chromatography and (1)H-nuclear magnetic resonance (NMR) to analyze vestimentiferans (vestimentum tissue) from unusual marine habitats. Species from deep hydrothermal vents were Riftia pachyptila of the East Pacific Rise (2,636 m) and Ridgeia piscesae of the Juan de Fuca Ridge (2,200 m). Species from cold hydrocarbon seeps were Lamellibrachia sp. and an unnamed escarpid species from subtidal sediment seeps (540 m) off Louisiana and Lamellibrachia barhami from bathyal tectonic seeps (1,800-2,000 m) off Oregon. Riftia were dominated by hypotaurine (152 mmol/kg wet wt), an antioxidant, and an unidentified solute with an NMR spectrum consistent with a methylamine. Ridgeia were dominated by betaine (N-trimethylglycine; 109 mmol/kg), hypotaurine (64 mmol/kg), and taurine (61 mmol/kg). The escarpids were dominated by taurine (138 mmol/kg) and hypotaurine (69 mmol/kg). Both Lamellibrachia populations were dominated by N-methyltaurine (209-252 mmol/kg), not previously reported as a major osmolyte, which may be involved in methane and sulfate metabolism. Trunk and plume tissue of the Oregon Lamellibrachia were nearly identical to vestimentum in osmolyte composition. The methylamines may also stabilize proteins against pressure; they were significantly higher in the three deeper-dwelling groups.
Although neutrophilic granulocytes are assumed to contribute to cartilage degradation during rheumatic diseases, there is still a discussion whether reactive oxygen species (ROS) or proteolytic enzymes that are both released by the neutrophils are most relevant to cartilage degradation. To gain further insight into these processes, an in vitro approach to study the interaction between the products of stimulated neutrophilic granulocytes and cartilage was used: Neutrophils from the blood of healthy volunteers were treated with different stimulators (e.g., Ca(2+) ionophores) in order to induce degranulation. Supernatants of neutrophils were afterward incubated with thin slices of pig articular cartilage. Some experiments were also performed in the presence of selected enzyme inhibitors. Supernatants of cartilage were subsequently assayed by one- and two-dimensional high-resolution proton NMR spectroscopy, and the content of soluble carbohydrates in the supernatant was additionally determined by biochemical methods. The selective inhibition of elastase decreased most significantly the extent of cartilage degradation, whereas all other inhibitors had much smaller effects. These results were additionally confirmed by measuring the effect of isolated elastase on articular cartilage in the absence and presence of different inhibitors. It is concluded that elastase released [EC 3.4.21.37] by neutrophils is the most relevant enzyme for cartilage degradation.
The Australian brush-tailed possum, Trichosurus vulpecula, is capable of producing a moderately concentrated urine, at least up to 1300 mOsm l(-1). Kidneys of adult animals fed in captivity on a normal diet with ready access to water were analysed. The inner medullary regions were found to have moderately high concentrations of sodium (outer medulla, 367+/-37; inner medulla 975+/-93 mmol kg(-1) dry wt.), chloride (outer medulla 240+/-21; inner medulla 701+/-23 mmol kg(-1) dry wt.) and urea (outer medulla, 252+/-62; inner medulla, 714+/-69 mmol kg(-1) protein). When the animals were fed on a 'wet diet', amounts of these substances in the outer medulla and cortex were reduced, although with the exception of urea these changes were not significant. There were highly significant changes in amounts of Na(+), Cl(-) and urea in the inner medulla (Na(+), 566+/-7; Cl(-), 422+/-9 mmol kg(-1) dry wt.; urea 393+/-84 mmol kg(-1) protein). Likewise, the inner medulla of animals fed a 'dry diet' with limited access to water showed highly significant increases in the same substances (Na(+), 1213+/-167; Cl(-), 974+/-137 mmol kg(-1) dry wt.; urea, 1672+/-98 mmol kg(-1) protein). Inositol was found in the outer medulla (224+/-90 mmol kg(-1) protein) and inner medulla (282 mmol kg(-1) protein) as was sorbitol (outer medulla, 62+/-20; inner medulla, 274+/-72 mmol kg(-1) protein). Both these polyols were reduced in amount in renal tissue from 'wet diet' animals, and increased in 'dry diet' animals, but the changes were not statistically significant. The methylamines, betaine and glycerophosphorylcholine (GPC), showed a similar pattern, but both were significantly elevated in the inner medulla of 'dry diet' animals (betaine 154+/-57 to 315+/-29 mmol kg(-1) protein; GPC 35+/-7 to 47+/-10 mmol kg(-1) protein). It was concluded that in this marsupial the concentrating mechanism probably functions in a similar way to that in higher mammals, and that the mechanism of osmoprotection of the medulla of the kidney involves the same osmolytes. However, the high ratio of betaine to GPC in the inner medulla resembles the situation in the avian kidney.
Acetonitrile extracts of ulcerated and control rat stomachs were studied by various NMR techniques in an attempt to understand how indomethacin, a common and powerful nonsteroidal antiinflammatory drug (NSAID), induces ulcers in the stomach. One- (1D) and two-dimensional (2D) NMR spectra of extracts of ulcerated and control stomachs revealed that glycolytic and Krebs cycle enzymes were partially inhibited in the ulcerated stomach as shown by the lactate/glucose ratio. The (total choline)/lactate ratio was also higher in the extract from the control stomach than in the ulcerated stomach. Glycerophosphoethanolamine and glycerophosphocholine concentrations were higher in the ulcerated stomach extract as compared with the control stomach extract. These results explain the gastrointestinal protective effect of D-glucose and Krebs cycle intermediates on NSAID-induced ulceration.
This study describes a chemometric analysis of 1H NMR spectra of canine plasma following oral single dose administrations of two food components (lycopene and oleuropein) and of two drug products, Fungoral (ketoconazole) and Adalat (nifedipine). Due to the high interday physiological variation, 1H NMR plasma data were first filtered (by applying orthogonal signal correction) and then subjected to principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA). A distinct discrimination was achieved between samples obtained with and without administration of xenobiotics using both techniques. According to distance to model criterion and as shown by the Cooman's plots there was no overlap between the four models which proved to be specific for each xenobiotic. Moreover, it was shown that this approach has the potential to recognize subtle variations in the metabolic profile even if the administered xenobiotic itself could not be detected in the canine plasma by conventional HPLC methods.
A procedure of nuclear magnetic resonance (NMR) urinalysis using pattern recognition is proposed for early detection of toxicity of investigational compounds in rats. The method is applied to detect toxicity upon administration of 13 toxic reference compounds and one nontoxic control compound (mianserine) in rats. The toxic compounds are expected to induce necrosis (bromobenzene, paracetamol, carbon tetrachloride, iproniazid, isoniazid, thioacetamide), cholestasis (alpha-naphthylisothiocyanate (ANIT), chlorpromazine, ethinylestradiol, methyltestosterone, ibuprofen), or steatosis (phenobarbital, tetracycline). Animals were treated daily for 2 or 4 days except for paracetamol and bromobenzene (1 and 2 days) and carbon tetrachloride (1 day only). Urine was collected 24 h after the first and second treatment. The animals were sacrificed 24 h after the last treatment, and NMR data were compared with liver histopathology as well as blood and urine biochemistry. Pathology and biochemistry showed marked toxicity in the liver at high doses of bromobenzene, paracetamol, carbon tetrachloride, ANIT, and ibuprofen. Thioacetamide and chlorpromazine showed less extensive changes, while the influences of iproniazid, isoniazid, phenobarbital, ethinylestradiol, and tetracycline on the toxic parameters were marginal or for methyltestosterone and mianserine negligible. NMR spectroscopy revealed significant changes upon dosing in 88 NMR biomarker signals preselected with the Procrustus Rotation method on principal component discriminant analysis (PCDA) plots. Further evaluation of the specific changes led to the identification of biomarker patterns for the specific types of liver toxicity. Comparison of our rat NMR PCDA data with histopathological changes reported in humans and/or rats suggests that rat NMR urinalysis can be used to predict hepatotoxicity.
The effect of the antitumor complex [Au(dppe)2]Cl (where dppe is Ph2P(CH2)2PPh2) on the overall metabolism of cultured mouse L1 210 leukemia cells was investigated by comparing 1H and 31P NMR spectra of perchloric acid extracts of cells incubated for 1 h in the presence and absence of 2 μm [Au(dppe)2]Cl. There were marked (ca. two-fold) increases in the levels of lactate and almost all detectable amino acids suggesting a drug-induced increase in the rate of glycolysis and inhibition of protein synthesis. The levels of taurine and phosphorylcholine were significantly decreased and 31P NMR spectra revealed a depletion of nucleoside triphosphates (NTP). The effect on nucleotide metabolism was investigated further by separating purine and pyrimidine nucleotides and precursors by anion-exchange HPLC. NTP levels were depleted by ca. 70–90% and there was a ca. three-to four-fold increase in nucleoside di- and monophosphates. The effect is postulated to be the result of uncoupling of mitochondrial oxidative phosphorylation. The Cu(I) complex [Cu(Ph2PCH-CHPPh2)2]Cl produced a similar effect on the cellular metabolism but was more potent. The water-soluble complex [CU(Ph2P(CH2)PEt2)2]Cl caused the accumulation of cellular amino acids at a concentration that did not significantly deplete ATP levels. © 1991 Academic Press. Inc.
High resolution 1H-NMR spectra were obtained from renal cortical slices, homogenates, isolated cells in suspension, and lysates. Experimental conditions for acquisition of high resolution spin-echo spectra are described and assignments of prominent peaks to various metabolites are given. The in situ activity of the highly active cortical enzyme prolidase was determined (1.8 nmoles/min/mg wet kidney wt) by exploiting the marked difference in the spectral features of glycyl-L-proline and that of the free amino acids. The results suggest that this NMR method will be applicable generally to the study of hydrolysis of peptides, as well as to other metabolic processes both in vitro and in vivo.
Reactions of cis- and trans-[PtCl2(NH3)2] with glutathione (GSH) inside intact red blood cells have been studied by 1H spin-echo nuclear magnetic resonance (NMR). Upon addition of trans-[PtCl2(NH3)2] to a suspension of red cells, there was a gradual decrease in the intensity of the resonances for free GSH, and new peaks were observed that were assignable to coordinated GSH protons in trans-[Pt(SG)Cl(NH3)2], trans-[Pt(SG)2(NH3)2], and possibly the S-bridged complex trans-[[NH3)2PtCl)2SG]+. Formation of trans-[Pt(SG)2(NH3)2] inside the cell was confirmed from the 1H NMR spectrum of hemolyzed cells, which were ultrafiltered to remove large protein molecules; the ABM multiplet of the coordinated GSH cys-beta CH2 protons was resolved using selective-decoupling experiments. Seventy percent of the total intracellular GSH was retained by the ultrafiltration membrane, suggesting that the mixed complex trans-[Pt(SG)(S-hemoglobin)(NH3)2] also is a major metabolite of trans-[PtCl2(NH3)2] inside red cells. The reaction of cis-[PtCl2(NH3)2] with intracellular GSH was slower; only 35% of the GSH had been complexed after a 4-hr incubation compared to 70% for the trans isomer. There was a gradual decrease in the intensity of the GSH 1H spin-echo NMR resonances, but no new peaks were resolved. This was interpreted as formation of high-molecular weight Pt:GSH and mixed GS-Pt-S(hemoglobin) polymers. By using a 15N-[1H] DEPT pulse sequence, we were able to study the reaction of cis-[PtCl2(15NH3)2] with red cells at concentrations as low as 1 mM. 15NH3 ligands were released, and no resonances assignable to Pt-15NH3 species were observed after a 12-hr incubation.
Broad resonances at 2.04 and 2.08 ppm in 500 MHz Hahn spin-echo 1H NMR spectra of human blood plasma are assigned to the N-acetyl groups of mobile carbohydrate side-chains (largely N-acetylglucosamine and N-acetylneuraminic acid) of glycoproteins such as alpha 1-acid glycoprotein. Their intensities in spin-echo spectra correlate with clinical conditions in which an elevation of the level of 'acute-phase' glycoproteins is expected, and so may be of value in the study of certain diseases.
The hydrolysis of NAD by the extracellular membrane-associated enzyme NAD glycohydrolase was shown to be readily followed in concentrated suspensions of human erythrocytes using 1H spin-echo nuclear magnetic resonance spectroscopy (NMR). The maximal rate of the reaction was determined and the inhibitory effect of nicotinamide was confirmed by direct NMR observation. In addition, arginine, ergothioneine and iodoacetate did not influence the reaction rate. 31P NMR analyses of reaction media from whole cells showed that no extraneous degradation of NAD occurred and the only phosphate-containing product was ADP-ribose.
Single-pulse and Hahn spin-echo 500 MHz 1H NMR spectra of human blood plasma and isolated chylomicrons, VLDL, LDL and HDL are reported. The comparison has enabled specific assignments to be made for the resonances of individual lipoproteins in the CH2 and CH3 (fatty acid), and NMe+3 (phospholipid choline head group) regions of the spectra of plasma (0.8-1.3 and approximately 3.25 ppm, respectively). Fasting, and freeze-thawing of plasma samples led to marked changes in the intensities and linewidths of lipid resonances. Analysis of lipid resonances in the spectra of plasma in terms of individual lipoproteins may shed new light on many conditions of clinical and biochemical interest.
Two-dimensional proton-proton correlated NMR spectra of concentrated urine from a subject who had ingested a 1-g dose of acetaminophen are reported. These "maps" provide a considerable simplification of the spectrum in comparison with conventional one-dimensional NMR spectra. In the present case, peaks for all the major acetaminophen metabolites, including the L-cysteinyl conjugate, can be unambiguously assigned.
The first application of inversion-recovery spin-echo proton nuclear magnetic resonance spectroscopy to the monitoring of reactions in rat brain preparations is presented. The initial report of the assignment of proton spin-echo nuclear magnetic resonance spectra from rabbit brain homogenates (C. R. Middlehurst et al., J. Neurochem. 42, 878–879, 1984) was used to assist in the assignment of spectra acquired from rat brain homogenates that were obtained from animals killed by cervical fracture or focussed microwave irradiation. Microwave-irradiated brains were divided into four major anatomical regions. Differences in metabolite levels were detected when spectra from fresh tissue and from various regions were compared. The in situ steady-state kinetics of prolidase in whole brain homogenate was determined.
The procedure relies on the spectral differences between enzyme substrates and reaction products. The concentration dependence of the rate of hydrolysis of glycyl-l-proline was describable by the Michaelis-Menten expression with a Michaelis constant of 1.90 mmol L-1 and a maximal velocity of 9.30 μmol min-1 mg-1 protein. The reactions catalysed by glutaminase and acetylcholinesterase in the brain were also monitored.
The kinetics of several metabolic reactions in intact human erythrocytes and in lysates were studied using 1H spin-echo and 13C nuclear magnetic resonance spectroscopy (NMR). The reactions monitored involved the following enzymes: (1) arginase, (2) glutathione reductase, (3) glutathione synthetase, (4) gamma-glutamyl cyclotransferase, (5) di- and tripeptidase, and (6) NAD-glycohydrolase; the first six enzymes are cytosolic whilst the latter is membrane associated. Detailed kinetics of the arginase reaction are given together with the rate of arginine transport into the cells.
Numerous low-Mr metabolites--including creatinine, citrate, hippurate, glucose, ketone bodies, and various amino acids--have been identified in 400- and 500-MHz proton nuclear magnetic resonance (1H NMR) spectra of intact human urine. The presence of many of these was related to the specific condition of the donors: humans in different physiological states (resting, fasting, or post-exercise) and pathological conditions (e.g., diabetes mellitus, cadmium-induced renal dysfunction). We have also monitored the metabolism of simple nonendogenous compounds (methanol and ethanol) and of acetaminophen. The pH-dependencies of the NMR chemical shifts of some urine components are reported. Our studies show that high-resolution 1H NMR spectroscopy provides a fast, simple method for "fingerprint" identification of urinary compounds. In some cases, analytes can be quantified by standard additions or by comparing integrated peak areas for the metabolites with those for creatinine. Determinations of creatinine by 1H NMR spectroscopy compared well with those by an independent chemical assay based on the Jaffé reaction.
Spin echo Fourier transform proton n.m.r. spectra of whole blood contain resonances from both erythrocytes and plasma. A large number of well-resolved signals from mobile protons of low-molecular-weight metabolites in plasma and serum have been identified. Spectra from the plasmas of eight animal species and commercial, quality control sera are compared. CaEDTA2- and MgEDTA2- resonances can be used for the simultaneous determination of EDTA-chelatable calcium and magnesium concentrations in intact plasma and other biological fluids. Cholesterol is too immobile to contribute to the spectra of intact plasma, but is readily estimated by n.m.r. in both its free and esterified forms after extraction into methanol.
Proton nuclear magnetic resonance (1H NMR) spectroscopy in conjunction with the inversion-recovery spin-echo pulse sequence was used to obtain spectra from rabbit brain homogenate. The instrumental parameters required for the acquisition of spectra together with the assignment of major peaks are given. The rationale and prospectus for the use of this technique for the study of neurochemistry is outlined.
1H spin-echo NMR spectroscopy has been used to show for the first time, to our knowledge, the presence of a phospholipase D activity in human erythrocytes. The enzyme is presumably intra-cellular and we have demonstrated that it is activated by a component of Krebs bicarbonate medium; most probably calcium ions. The existence of a phospholipase in human erythrocytes allows a simple explanation for the choline accumulation that occurs in these cells of manic-depressive and other patients receiving lithium carbonate therapy.
Numerous low-Mr metabolites--including creatinine, citrate, hippurate, glucose, ketone bodies, and various amino acids--have been identified in 400- and 500-MHz proton nuclear magnetic resonance (1H NMR) spectra of intact human urine. The presence of many of these was related to the specific condition of the donors: humans in different physiological states (resting, fasting, or post-exercise) and pathological conditions (e.g., diabetes mellitus, cadmium-induced renal dysfunction). We have also monitored the metabolism of simple nonendogenous compounds (methanol and ethanol) and of acetaminophen. The pH-dependencies of the NMR chemical shifts of some urine components are reported. Our studies show that high-resolution 1H NMR spectroscopy provides a fast, simple method for "fingerprint" identification of urinary compounds. In some cases, analytes can be quantified by standard additions or by comparing integrated peak areas for the metabolites with those for creatinine. Determinations of creatinine by 1H NMR spectroscopy compared well with those by an independent chemical assay based on the Jaffé reaction.
Recent developments in high-resolution 1H NMR allow biological samples to be analyzed for certain low mol. wt. components with little or no sample pre-treatment. The 1H NMR methods require only small sample volumes (0.3 ml), are non-destructive and usually give qualitative results within a few minutes if adequate steps are taken to reduce the intensity of the solvent water signal with an appropriate secondary irradiation field or pulse sequence. Notably, preselection of instrumental conditions to observe different classes of compound is not necessary, e.g. when studying compounds whose metabolic and excretory mechanisms are poorly understood.
The latter is the case for N-methylformamide (NMF), an experimental anti-tumour agent, but not for paracetamol. 1H NMR has been applied to the detection and quantitation of urinary metabolites of these drugs. For paracetamol, the free drug and major metabolites (glucuronide, sulphate, cysteinyl and N-acetylcysteinyl conjugates) were all detected in untreated urine, and there was quantitative agreement with HPLC results. However, two-dimensional (2-D) NMR methods were required to separate the aramatic resonances of the cysteinyl conjugate and the free drug. For NMF, 1H NMR measurements led to detection and identification of previously unknown metabolites including formic acid and a novel N-acetylcysteinyl derivative (N-acetyl-S-[N-methyl-carbamoyl]cysteine) as well as the parent compound and methylamine. Signals for various excreted compounds of endogenous origin are also manifest in 1H NMR spectra of urine, so that information on the toxicological effects of drugs and xenobiotics can be obtained simultaneously with data on their metabolism.
Human erythrocytes were subjected to oxidative stress using arsenicals. The study is a model for thiol-related redox processes observed in the etiology of diseases such as rheumatoid arthritis. Spin-echo NMR spectroscopy of the living cell was used to monitor the cellular biochemistry. Oxidation of glutathione and the first demonstrated response of ergothioneine to a chemical stimulus in a cell were observed. The reversible nature of the ergothioneine response is interpreted as an environmental rather than a chemical change. © 1988 Academic Press, Inc.
The products of the reactions of cis- and trans-[PtCl2(NH3)2] with reduced glutathione (GSH) in phosphate-buffer (pH 7) have been characterized by 1H, 13C, 15N and 195Pt NMR, with the aim of identifying the likely metabolites of the complexes with intracellular GSH. The final product of the reaction of trans-[PtCl2(NH3)2] with GSH was the bis-substituted complex trans-[Pt(SG)2(NH3)2] in which glutathione was coordinated exclusively via the S atom. Trans-[Pt(SG)Cl(NH3)2] was formed in the first step and reacted either with GSH to form trans-[Pt(SG)2(NH3)2], or with trans-[PtCl2(NH3)2] to give the S-bridged complex trans-[{(NH3)2PtCl}2SG]+. This reacted with GSH regenerating trans-[Pt(SG)Cl(NH3)2]. By the use of a 15N-{1H} DEPT pulse sequence, 15N NMR was used to follow the transient intermediates of the reaction of cis-[PtCl2(15NH3)2] with GSH. The mono-substituted complex cis-[PtCl(SG)(15NH3)2] was formed first, but several other species containing a 15NH3-Pt-S linkage were present within a few minutes of starting the reaction. 15NH3 ligands began to be released within 10 min and the final product contained no coordinated 15NH3. The 1H, 13C, and 195Pt NMR spectra of the final product were consistent with a high-molecular weight polymer with a 1:2 Pt:GSH stoichiometry, in which coordination was almost exclusively via the S atom, but with several different Pt-S and Pt-S-Pt environments.
The purpose of this article is to review some of the NMR techniques used to measure ¹H NMR spectra of human plasma and red blood cells. The ¹H NMR spectroscopy of plasma and red blood cells of interest because reported studies indicate that information relevant to biochemical and clinical applications can be obtained by ¹H NMR.
Normal sheep erythrocytes as well as glutathione- (GSH-) deficient and arginase-deficient sheep erythrocytes have been characterized by 1H nuclear magnetic resonance spectroscopy. The GSH deficiency is a result of defective amino acid transport (lesion 1), diminished γ-glutamylcysteine synthetase activity (lesion 2), or both (lesions (1 + 2)). 1H-NMR spectra of normal sheep erythrocytes are similar to those for human erythrocytes, and consist of resonances from a number of small intracellular molecules, including GSH. In contrast, the resonances for GSH in the GSH-deficient erythrocytes are much weaker, and strong resonances are observed for lysine, threonine and ornithine or arginine, depending on the arginase activity, in erythrocytes with lesion 1 and lesions (1 + 2). A comparison of the intensity of GSH resonances in spectra for normal and GSH-deficient erythrocytes with GSH levels determined spectrophotometrically following reaction with the nonspecific thiol reagent 5,5′-dithiobis(2-nitrobenzoate) (DTNB) indicates that either not all of the GSH determined with Ellman's reagent is free and observable by 1H-NMR or that not all of the thiol determined by Ellman's reagent is GSH. If the latter is the case, the GSH levels determined with Ellman's reagent for erythrocytes with lesions (1 + 2) are most affected, which might account for their high susceptibility to oxidative stress.
The elevated levels of brain histidine in histidinaemic mice are detected in vivo by 1H NMR at 8.5 and 1.9 T. The concentrations determined from the in vivo spectra correlate well with subsequent analytical determinations. This technique is discussed in relation to monitoring phenylalaninc in humans with phenylketonuria. © 1986 Academic Press, Inc.
Proton nuclear magnetic resonance (1H NMR) spectroscopy in conjunction with the inversion-recovery spin-echo pulse sequence was used to obtain spectra from rabbit brain homogenate. The instrumental parameters required for the acquisition of spectra together with the assignment of major peaks are given. The rationale and prospectus for the use of this technique for the study of neurochemistry is outlined.
Studies on the role of red blood cell organic phosphates in regulating oxygen transport and hemoglobin function in various species have emanated from several laboratories in the past few years. These data reveal that the organic phosphate composition of erythrocytes is certainly more diverse between classes of animals than previously recognized, that the kind and concentration of red cell organic phosphate modulator changes during development of the species and that the role of organic phosphates in modulating hemoglobin function is variable. For example in birds we believe that the changes in amounts and distribution of the intraerythrocytic organic phosphates account for the sudden changes in whole blood oxygen affinity during development of the embryo and young bird as well as in the mature bird.
In the loggerhead and green sea turtle it appears that organic phosphate modulators regulate whole blood oxygen affinity during embryonic development but it is unlikely that whole blood oxygen affinity is controlled by organic phosphate modulation of hemoglobin function in the adult turtle.
From the data now available on air-breathing fishes, it appears reasonable to consider that whole blood oxygen affinity may be regulated by a combination of organic phosphates that is the relative concentrations of the primary regulator (inositol-P2 2,3-DPG, or inositol P5) may be rather stable whereas the nucleotide triphosphate (ATP and GTP) concentrations may fluctuate depending upon physiological demands upon the animal for oxygen.
A high concentration of a basic unidentified amino compound was found in the blood of rats. It was isolated and identified as N epsilon,N epsilon,N epsilon-trimethyllysine by paper chromatography, thin-layer chromatography, high-performance liquid chromatography and amino acid analyzer. It was localized exclusively in red blood cells in the blood of rats. Free trimethyllysine was also determined in the liver, kidney, spleen, brain, muscle, heart and testis of rat. The concentration of free trimethyllysine in red blood cells was more than 10-times as high as that in the other tissues. This compound in red blood cells was found in different species of animals. The relationship between this free trimethyllysine and carnitine was discussed.
Nuclear magnetic resonance (NMR) studies of extracts have proven to be a powerful window onto the intracellular machinery of cells and tissues. The major advantages of in vitro 1H-NMR, namely chemical preservation, simultaneous detection, identification, and quantitation of compounds, and sensitivity to a large variety of classes of compounds, are employed in this study to characterize the metabolic course of mitogen-stimulated proliferation of human peripheral lymphocytes. A reliable method to quantitate amino acids, metabolic intermediates, soluble membrane lipid precursors, and purine, pyridine and pyrimidine nucleotides is presented, using samples as small as 30 mg wet weight. A total of 53 substances were detected in lymphocytes and other blood cells. During the course of lymphocyte culture, changes in intracellular concentrations of lactate, taurine, inositol and nucleotides, including NAD, IMP and high-energy phosphates, were especially marked. 1H-NMR compares favorably to 31P-NMR and to HPLC, and is especially attractive in light of expectations for future in vivo application.
Localized proton NMR spectroscopy at 1.5 T using stimulated echoes has been applied to study metabolic alterations in the postischemic phase of patients with acute cerebral infarction. A complete depletion of N-acetyl aspartate in the area of infarction has been observed in a patient studied 4 days after stroke. This finding was paralleled by a dramatic increase in the concentration of lactic acid to about 16 mM within the lesion, indicating continued anaerobic glycolysis. The diluting effect of the edema has been estimated to reduce average metabolite concentrations by about a factor of 3.
Proton nuclear magnetic resonance (NMR) spectroscopy was used to follow glucose metabolism in Crithidia luciliae. Parasites were grown aerobically and anaerobically in culture, with glucose as the major carbon source and 1H NMR spectra were acquired for the cell free medium. The 1H NMR resonances of metabolites utilised and produced during cell growth were identified by difference spectroscopy, and quantitated from standard curves using 3-trimethylsilyl propionate-2,2,3,3-d4 sodium salt as an internal standard. The major metabolites produced by C. luciliae grown aerobically on 8 mM glucose were succinate, pyruvate, acetate and ethanol, in final concentrations in the media when the cells entered stationary phase of 8.5 +/- 0.5, 5.0 +/- 0.3, 2.1 +/- 0.2 and 2.5 +/- 0.6 mM, respectively. The production of succinate and pyruvate, but not acetate and ethanol, followed closely the growth curve of the parasites. Succinate was also measured enzymically and glucose using an autoanalyser. In both cases the results correlated well with the NMR data. The amounts of end products formed were greater than could be accounted for by the utilisation of glucose or any other metabolite observable in the 1H NMR spectra. There was approximately one extra atom of carbon for each molecule of succinate formed, supporting the view that succinate is produced via phosphoenolpyruvate carboxykinase and carbon dioxide fixation. Anaerobically the same major metabolites were produced, but with a decreased ratio of succinate to acetate and ethanol. The formation of glycerol from glucose was not observed under these conditions.
Isolated enterocytes prepared from chicken small intestine were studied by proton nuclear magnetic resonance (NMR). Perchloric acid extracts of cells were prepared from duodenum, jejunum, and ileum, and concentrations of 19 different metabolites were determined after the virtually complete assignment of all peaks in the aliphatic region of the spectrum. High concentrations of serine ethanolamine phosphodiester (SEP), creatine, taurine, and acidic amino acids were found in all segment extracts. Relatively high concentrations of SEP (approximately 12 mM) and acidic amino acids (approximately 3.5 mM) were found in the ileum, whereas creatine (3 mM) and lactate (6.5 mM) were found at higher concentrations in jejunum. Taurine (approximately 8 mM), choline (0.5 mM), and betaine (approximately 0.5 mM) were evenly distributed throughout the segments. Fasting (40 h) led to substantial increases in the concentrations of pyruvate, succinate, SEP, and taurine, while neutral hydrophobic amino acid concentrations fell appreciably. The significance of the findings is discussed, and the possible contributions of SEP, taurine, and choline to membrane lipid homeostasis are considered.
We have shown that with state-of-the-art NMR instrumentation and some simple NMR techniques, high-resolution 1H NMR spectra can easily be measured for small molecules in plasma and intact red blood cells. Although such measurements are not yet used routinely in clinical laboratories, results in the literature demonstrate that these techniques offer unique advantages for clinical measurements. Their value in elucidating the details of biochemical processes in intact red blood cells is already well established. These NMR techniques can also be used to study other biological fluids and cellular systems.
Human erythrocytes were subjected to oxidative stress using arsenicals. The study is a model for thiol-related redox processes observed in the etiology of diseases such as rheumatoid arthritis. Spin-echo NMR spectroscopy of the living cell was used to monitor the cellular biochemistry. Oxidation of glutathione and the first demonstrated response of ergothioneine to a chemical stimulus in a cell were observed. The reversible nature of the ergothioneine response is interpreted as an environmental rather than a chemical change.
Frog sartorius and gastrocnemius muscles were perifused at 20 degrees C, the intracellular pH (pHi) and the concentration of phosphocreatine were determined in the resting muscle by 1H-NMR spectroscopy at 470 MHz; values of pHi = 7.31 +/- 0.05 (n = 7) and concentration of phosphocreatine = 20.4 +/- 1.1 mumol/g wet wt. (n = 6) were found. The hydrolysis of phosphocreatine and the simultaneous increase in lactate upon perifusion with 10 mM caffeine (in Ringer's solution) was followed with a time resolution of 1 min. Lactate increased at a rate of 1.0 mumol/g per min, but no pHi change was recorded during the time monitored. The lower limit for the buffering capacity of the muscle cytosol was estimated to be 16.7 mumol/g muscle per pH unit from the uncertainty in pHi determination (+/- 0.03 pH units) and from the amount of lactate produced and phosphocreatine hydrolyzed. Changes in pHi, lactate concentration and fatty acyl chain intensity were monitored by 1H-NMR spectroscopy at 361 MHz in ischemic rat skeletal muscle, excised and stored at 20 degrees C. The resonances in the 1H-NMR spectrum of a human skeletal muscle perchloric acid extract are reported and tentatively assigned.
1H-NMR was applied to the study of metabolites in human saliva specimens. Acetate, lactate, ethanol, glucose and some other substances were simultaneously identified and quantitated from the 1H-NMR spectra of saliva specimens treated with D2O. These experiments demonstrated the value of 1H-NMR as an analytical method in the field of forensic science and clinical pathology and toxicology.
High-resolution proton NMR spectra from nine samples of human aqueous humour extracted from the anterior chamber of the eye are reported. They were obtained within the order of 45 min from small volumes (ca 0.3 ml) of sample with minimal pretreatment and the method was non-destructive. Metabolites detected included acetate, acetoacetate, alanine, ascorbate, citrate, creatine, formate, glucose, glutamine-glutamate, beta-hydroxybutyrate, lactate, threonine and valine. Concentrations of lactate, valine, alanine and acetate were determined. These novel metabolite profiles may be of value in the study of clinical disorders.
High-resolution proton magnetic resonance spectroscopy was used to analyze human cerebrospinal fluid obtained from patients with several neurological problems. The major metabolites measured included glucose, lactate, glutamine, citrate, inositol, acetate, creatine, creatinine, beta-hydroxybutyrate, alanine, and pyruvate. A drug vehicle, propylene glycol, was also measured. Alterations in the cerebrospinal fluid of these metabolites provided information concerning metabolism of the brain. Magnetic resonance spectroscopy offered a simple and rapid means of assessing these and other exogenous and endogenous compounds in diseases affecting the nervous system.
High-resolution 1H nuclear magnetic resonance (1H-n.m.r.) spectroscopy at 300 MHz has been used to monitor the excretion in human urine of the drug metronidazole and its metabolites. One or more resonances from all of the major metabolites are resolved in spectra of whole urine, and can be used to quantify excretion of the parent drug and its metabolites. The spectra also contain resonances from a large number of endogenous metabolites, many of which are readily identified. 1H n.m.r. should be useful in monitoring, simultaneously and non-destructively, the urinary excretion of drugs and drug metabolites, as well as endogenous compounds in either normal or disease states.
High resolution proton nuclear magnetic resonance (NMR) spectra of normal and diseased human muscle extracts were recorded at 470 MHz. Resonances from lactic acid, creatine, glucose, ribose, purine and pyrimidine bases were identified. The longitudinal relaxation times of these resonances were determined to allow quantitation of muscle metabolites. With aid of a standardized reference capillary, inserted into the NMR tube containing the muscle extracts, the lactic acid and total creatine content of the extracts was determined. After 5 h of incubation at 37 degrees C, normal muscles contained on average 103 mumol lactic acid and 36 mumol creatine/173 mg of noncollagenous protein, equivalent to 1.0 g of fresh muscle. The lactic acid and creatine contents decreased slightly in scoliosis and idiopathic scoliosis and they decreased significantly in cerebral palsy. In an extract of a patient whose illness was diagnosed as 'scoliosis' no creatine was present, and in an extract of a patient with unknown diagnosis the creatine content was reduced to 2 mumol/173 mg of noncollagenous protein. The short time (1.7 sec to 6.5 min) and the small amount of tissue (300 mg) needed for an analysis add to the potential of proton NMR as a new technique for the characterization of muscular diseases.
1H NMR spectra of intact frog, and chicken skeletal muscles, were recorded at 470 MHz with the Plateau and Guéron pulse sequence for the suppression of water [(1982) J. Am. Chem. Soc. 104, 7310]. Only a few transients were required to resolve the resonances from the protons of muscle metabolites. The previously unobserved exchangeable protons of muscles were also recorded and thereby phosphocreatine and creatine could be measured simultaneously. During aging of dissected frog muscle, changes in levels of phosphocreatine, creatine and lactic acid, and the decrease in the intracellular pH were followed by 1H NMR.
We have demonstrated that magnetic susceptibility effects can strongly influence nuclear magnetic resonance spectra, particularly in cells, and lead to spurious line broadening and chemical shifts which will result in false conclusions about molecular motion and intracellular pH and equilibrium constants. Three specific instances of a general phenomenon resulting from the difference in magnetic susceptibility inside and outside deoxygenated red cells have been examined. We find that at 95 MHz the resonance of 19F covalently bound to both hemoglobin and glutathione as the trifluoroacetonyl compound undergoes substantial line broadening in deoxygenated red cells. Line broadening is not observed in oxygenated solutions or oxygenated cells nor is it observed in deoxygenated hemoglobin solutions. The broad lines in the deoxy red cell can be narrowed by matching the magnetic susceptibility of the suspending medium to that of the cell interior by adding suitable amounts of a paramagnetic metal ion such as manganese or dysprosium to the suspending medium. Similarly, we have observed line broadening of the 31P resonance of 2,3-diphosphoglyceric acid which occurs only in deoxygenated red cells and has a field dependence greater than the first power. This line broadening does not occur in oxygenated solutions or oxygenated cells nor does it occur in deoxygenated hemoglobin solutions. Again, the broadened lines in the deoxygenated cells can be narrowed by matching the magnetic susceptibility. Because the line-broadening effects reported here do not depend on the nucleus, the chemical nature of the molecule, or the molecular size, they cannot be attributed to specific chemical interactions or interactions with the cell membrane. Because they can be eliminated by matching the internal and external magnetic susceptibility, we attribute the observed broadening to magnetic susceptibility differences. Differences in magnetic susceptibility will also result in systematic displacement of the chemical shift of resonances arising inside the cell which may either add to or cancel effects due to biochemical interactions. We show that the appropriate corrections can be estimated but that due to the presence of magnetic field gradients use of internal references may be necessary.
1H-nuclear magnetic resonance (NMR) was applied to the study of metabolites in rat organs and tissues. From 1H-NMR spectra of D2O extracts of various organs and tissues, lactate, pyruvate, and some other substances were simultaneously identified and quantitated. On the basis of the results, the use of 1H-NMR for estimating post-mortem time was suggested.
High resolution 1H NMR spectra of perfused rat hearts have been obtained under normoxic, ischemic and hypoxic conditions. Several myocardial metabolites including taurine, carnitine, lactate and tissue glycerides are detected in the 1H NMR spectra. Changes in oxygen availability induce perturbations in the levels of some metabolites, in particular, lactate. Experiments with fasted rats and with substrate-free perfusion suggest that the glycerides detected in 1H spectra are metabolically mobilizable but have a slow rate of turnover. These results demonstrate that utility of 1H NMR in monitoring myocardial metabolism.
Improvements in nuclear magnetic resonance (NMR) technology are generating an expanding variety of medical applications. In this investigation I have used high-field proton NMR to identify and quantity endogenous and ingested substances in human serum. After addition of a small amount of 2H2O and a reference compound to a 0.4-mL specimen, spectra were recorded for 3 min in Fourier-transform mode, with use of presaturation to suppress the extremely intense H2O peak. Compounds detected at clinically significant concentrations include glucose, alcohols, acetone, organic acids, and salicylate. Less than 1 mmol/L of some of these substances could be detected. For serum containing 20--500 mg of added methanol per liter, peak area was a linear function of concentration (r = 0.998). High-field proton NMR, despite the drawback of expensive, sophisticated instrumentation, offers some unique advantages for clinical chemistry: it permits rapid, specific, nondestructive measurement of several compounds simultaneously, including some that may be inconvenient to measure by conventional means.
Proton nuclear magnetic resonance of intact Friend leukemia cells was used to analyze their erythroid-like differentiation. The technique, which requires only 10(3) to 10(9) cells and approximately 2 minutes for acquisition of each spectrum, demonstrated the occurrence of many signal changes during differentiation. With cell extracts, 64 signals were assigned to 12 amino acids and 19 other intermediary metabolites, and a dramatic signal change was attributed to a fourfold increase in cytoplasmic phosphorylcholines.