Resonance Spectroscopy Of Plasma Research Articles

Quantitative In-Vitro Diagnostic NMR Spectroscopy for Lipoprotein and Metabolite Measurements in Plasma and Serum: Recommendations for Analytical Artifact Minimization with Special Reference to COVID-19/SARS-CoV-2 Samples.

Quantitative nuclear magnetic resonance (NMR) spectroscopy of blood plasma is widely used to investigate perturbed metabolic processes in human diseases. The reliability of biochemical data derived from these measurements is dependent on the quality of the sample collection and exact preparation and analysis protocols. Here, we describe systematically, the impact of variations in sample collection and preparation on information recovery from quantitative proton (1H) NMR spectroscopy of human blood plasma and serum. The effects of variation of blood collection tube sizes and preservatives, successive freeze–thaw cycles, sample storage at −80 °C, and short-term storage at 4 and 20 °C on the quantitative lipoprotein and metabolite patterns were investigated. Storage of plasma samples at 4 °C for up to 48 h, freezing at −80 °C and blood sample collection tube choice have few and minor effects on quantitative lipoprotein profiles, and even storage at 4 °C for up to 168 h caused little information loss. In contrast, the impact of heat-treatment (56 °C for 30 min), which has been used for inactivation of SARS-CoV-2 and other viruses, that may be required prior to analytical measurements in low level biosecurity facilities induced marked changes in both lipoprotein and low molecular weight metabolite profiles. It was conclusively demonstrated that this heat inactivation procedure degrades lipoproteins and changes metabolic information in complex ways. Plasma from control individuals and SARS-CoV-2 infected patients are differentially altered resulting in the creation of artifactual pseudo-biomarkers and destruction of real biomarkers to the extent that data from heat-treated samples are largely uninterpretable. We also present several simple blood sample handling recommendations for optimal NMR-based biomarker discovery investigations in SARS CoV-2 studies and general clinical biomarker research.

A Novel Metabolic Signature To Predict the Requirement of Dialysis or Renal Transplantation in Patients with Chronic Kidney Disease.

Identification of chronic kidney disease patients at risk of progressing to end-stage renal disease (ESRD) is essential for treatment decision-making and clinical trial design. Here, we explored whether proton nuclear magnetic resonance (NMR) spectroscopy of blood plasma improves the currently best performing kidney failure risk equation, the so-called Tangri score. Our study cohort comprised 4640 participants from the German Chronic Kidney Disease (GCKD) study, of whom 185 (3.99%) progressed over a mean observation time of 3.70 ± 0.88 years to ESRD requiring either dialysis or transplantation. The original four-variable Tangri risk equation yielded a C statistic of 0.863 (95% CI, 0.831-0.900). Upon inclusion of NMR features by state-of-the-art machine learning methods, the C statistic improved to 0.875 (95% CI, 0.850-0.911), thereby outperforming the Tangri score in 94 out of 100 subsampling rounds. Of the 24 NMR features included in the model, creatinine, high-density lipoprotein, valine, acetyl groups of glycoproteins, and Ca2+-EDTA carried the highest weights. In conclusion, proton NMR-based plasma fingerprinting improved markedly the detection of patients at risk of developing ESRD, thus enabling enhanced patient treatment.

Lipoprotein particles and incident type 2 diabetes in the multi-ethnic study of atherosclerosis.

OBJECTIVEIn the Multi-Ethnic Study of Atherosclerosis (MESA), we evaluated associations of baseline levels of a lipoprotein-based insulin resistance (IR) index (LP-IR), IR-related lipoprotein particles, mean particle sizes, and lipids, with incident type 2 diabetes, independent of confounders, glucose, insulin, and HOMA-IR.RESEARCH DESIGN AND METHODSAmong 5,314 adults aged 45–84 years without baseline diabetes or cardiovascular disease, 656 cases of diabetes were identified during a mean follow-up of 7.7 years. Lipoprotein particle concentrations, size, and LP-IR were determined by nuclear magnetic resonance spectroscopy of stored baseline plasma. Potential effect modification, by race/ethnicity, sex, baseline use of lipid-lowering medications or hormone therapy, or glucose strata (<90, 90–99, and ≥100 mg/dL), was also evaluated.RESULTSHigher levels of LP-IR, large VLDL particles (VLDL-P), small LDL particles, triglycerides (TG), and TG–to–HDL cholesterol (HDL-C) ratio and lower levels of large HDL particles, smaller HDL and LDL size, and larger VLDL size were significantly associated with incident diabetes adjusted for confounders and glucose or insulin. These also were similar by race/ethnicity, sex, and treatment group. Associations were similar for LP-IR, large VLDL-P, mean VLDL size, TG, and TG–to–HDL-C ratio; they persisted for LP-IR, large VLDL-P, or mean VLDL size adjusted for HOMA-IR or TG–to–HDL-C ratio and glucose but not for the TG–to–HDL-C ratio adjusted for LP-IR or for HOMA-IR or insulin if adjusted for LP-IR and glucose.CONCLUSIONSAmong ethnically diverse men and women, LP-IR, large VLDL-P, large VLDL size, TG, and TG–to–HDL-C ratio were associated with incident diabetes independent of established risk factors, glucose, insulin, or HOMA-IR, as well as the use of lipid-lowering medications or hormone therapy.

NMR‐based metabolomics and breath studies show lipid and protein catabolism during low dose chronic T1AM treatment

Objective3-iodothyronamine (T1AM), an analog of thyroid hormone, is a recently discovered fast-acting endogenous metabolite. High single dose treatments of T1AM have produced rapid short-term effects, including a reduction of body temperature, bradycardia, and hyperglycemia in mice.Design and MethodsThe present study monitored the effect of daily low doses of T1AM (10mg/Kg) for eight-days on weight loss and metabolism in spontaneously overweight mice. The experiments were repeated twice (n=4). Nuclear magnetic resonance (NMR) spectroscopy of plasma and real-time analysis of exhaled 13CO2 in breath by cavity ringdown spectroscopy (CRDS) were used to detect T1M-induced lipolysis.ResultsCRDS detected increased lipolysis in breath shortly after T1AM administration that was associated with a significant weight loss but independent of food consumption. NMR spectroscopy revealed alterations in key metabolites in serum: valine, glycine, and 3-hydroxybutyrate, suggesting that the subchronic effects of T1AM include both lipolysis and protein breakdown. After discontinuation of T1AM treatment, mice regained only 1.8% of the lost weight in the following two weeks, indicating lasting effects of T1AM on weight maintenance.ConclusionsCRDS in combination with NMR and 13C-metabolic tracing constitute a powerful method of investigation in obesity studies for identifying in vivo biochemical pathway shifts and unanticipated debilitating side effects.

31P and 1H Nuclear Magnetic Resonance Spectroscopy of Blood Plasma in Female Patients with Preeclampsia

Objective: Using 31P and 1H magnetic resonance spectroscopy to measure phosphorus- and hydrogen-containing metabolites, this study aimes to investigate whether or not women with preeclampsia have detectable systemic abnormalities concerning certain components of the blood plasma. Methods: Plasma was obtained from two groups of women: Group 1 with preeclampsia (n=10) and Group 2, as a control group with no complications during pregnancy (n=10). Plasma analysis were performed using in-vitro 31P and 1H nuclear magnetic resonance spectroscopy. Results: 31P nuclear magnetic resonance (NMR) spectra showed significantly higher levels of lysophosphatidylcholine 1 in the plasma of the patients in Group 1, along with significantly decreased levels of lysophosphatidylcholine 2 and phosphatidylinositol. However, the total amount of phospholipids did not differ significantly between the groups. In addition, the 1H NMR spectra showed a significantly lower level of HDL in samples from Group 1, and a trend towards higher plasma levels of VLDL 2 and LDL 2 in the same group. Conclusion: This study supports the theory that preeclampsia is a disorder in phospholipid metabolism in which malfunctioning of cellular membranes seems to play a major pathogenic role.

PTU-045 Proton nuclear magnetic resonance spectroscopy of plasma in patients with cirrhosis correlates with arterial ammonia but not grade of hepatic encephalopathy

IntroductionDiagnosis of overt hepatic encephalopathy (HE) in patients with cirrhosis often relies on subjective clinical examination. Arterial ammonia correlates poorly with HE grades and a blood marker for diagnosis and...

Metabolic effects of albumin therapy in acute lung injury measured by proton nuclear magnetic resonance spectroscopy of plasma: A pilot study*

Improved means to monitor and guide interventions could be useful in the intensive care unit. Metabolomic analysis with bioinformatics is used to understand mechanisms and identify biomarkers of disease development and progression. This pilot study evaluated plasma proton nuclear magnetic resonance spectroscopy as a means to monitor metabolism following albumin administration in acute lung injury patients. This study was conducted on plasma samples from six albumin-treated and six saline-treated patients from a larger double-blind trial. The albumin group was administered 25 g of 25% human albumin in 0.9% saline every 8 hrs for a total of nine doses over 72 hrs. A 0.9% concentration of saline was used as a placebo. Blood samples were collected immediately before, 1 hr after, and 4 hrs after the albumin/saline administration for the first, fourth, and seventh doses (first dose of each day for 3 days). Samples were analyzed by proton nuclear magnetic resonance spectroscopy, and spectra were analyzed by principal component analysis and biostatistical methods. None. After 1 day of albumin therapy, changes in small molecules, including amino acids and plasma lipids, were evident with principal component analysis. Differences remained 3 days after the last albumin administration. Analysis of data along with spectra from healthy controls showed that spectra for patients receiving albumin had a trajectory toward the spectra observed for healthy individuals while those of the placebo controls did not. The data suggest that metabolic changes detected by proton nuclear magnetic resonance spectroscopy and the bioinformatics tool may be a useful approach to clinical research, especially in acute lung injury.

Individual variation in macronutrient regulation measured by proton magnetic resonance spectroscopy of human plasma

Proton nuclear magnetic resonance ((1)H-NMR) spectroscopy of plasma provides a global metabolic profiling method that shows promise for clinical diagnostics. However, cross-sectional studies are complicated by a lack of understanding of intraindividual variation, and this limits experimental design and interpretation of data. The present study determined the diurnal variation detected by (1)H NMR spectroscopy of human plasma. Data reduction methods revealed three time-of-day metabolic patterns, which were associated with morning, afternoon, and night. Major discriminatory regions for these time-of-day patterns included the various kinds of lipid signals (-CH(2)- and -CH(2)OCOR), and the region between 3 and 4 ppm heavily overlapped with amino acids that had alpha-CH and alpha-CH(2). The phasing and duration of time-of-day patterns were variable among individuals, apparently because of individual difference in food processing/digestion and absorption and clearance of macronutrient energy sources (fat, protein, carbohydrate). The times of day that were most consistent among individuals, and therefore most useful for cross-sectional studies, were fasting morning (0830-0930), postprandial afternoon (1430-1630), and nighttime samples (0430-0530). Importantly, the integrated picture of metabolism provided by (1)H-NMR spectroscopy of plasma suggests that this approach is suitable to study complex regulatory processes, including eating patterns/eating disorders, upper gastrointestinal functions (gastric emptying, pancreatic, biliary functions), and absorption/clearance of macronutrients. Hence, (1)H-NMR spectroscopy of plasma could provide a global metabolic tolerance test to assess complex processes involved in disease, including eating disorders and the range of physiological processes causing dysregulation of energy homeostasis.

Experimental Metabonomic Model of Dietary Variation and Stress Interactions

Stress in the form of moderate periods of maternal separation of newborn rats has been postulated to cause permanent changes in the central nervous system and diseases in later life. It is also considered that dietary supplementation with long chain polyunsaturated fatty acids (LC-PUFAs) can potentially ameliorate the effects of stress. The metabolic consequences of early life maternal separation stress were investigated in rats (2-14 days after birth), either alone or in combination with secondary acute water avoidance stress at 3-4 months of age. The effect of a LC-PUFA-enriched dietary intervention in stressed animals was also assessed. Systematic changes in metabolic biochemistry were evaluated using 1H nuclear magnetic resonance spectroscopy of blood plasma and multivariate pattern recognition techniques. The biochemical response to stress was characterized by decreased levels of total lipoproteins and increased levels of amino acids, glucose, lactate, creatine, and citrate. Secondary acute water avoidance stress also caused elevated levels of O-acetyl glycoproteins in blood plasma. LC-PUFAs dietary enrichment did not alter the metabolic response to stress, but did result in a modified lipoprotein profile. This work indicates that the different stressor types resulted in some common systemic metabolic responses that involve changes in energy and muscle metabolism, but that they are not reversible by dietary intervention.

A DECREASE IN 1 H NUCLEAR MAGNETIC RESONANCE SPECTROSCOPICALLY DETERMINED CITRATE IN HUMAN SEMINAL FLUID ACCOMPANIES THE DEVELOPMENT OF PROSTATE ADENOCARCINOMA

Because human prostatic fluid contributes almost 50% of the volume of seminal plasma and this fluid contains unique prostatic metabolites such as citrate, which are markedly altered during tumorigenesis, we investigated high resolution H nuclear magnetic resonance (NMR) spectroscopy of unprocessed human seminal plasma as a rapid, noninvasive diagnostic tool for prostate adenocarcinoma. Semen and prostatic massage samples from control and tumor bearing subjects were stored frozen at -20C and thawed prior to water suppressed NMR analysis. We found that freezing produced no significant alterations in the semen NMR spectra. Quantitative NMR spectroscopy was performed by first calibrating the water suppression data acquisition sequence with a series of standard samples containing known amounts of citrate within the physiological range. Well resolved citrate resonances from the seminal plasma of 3 control subjects with prostate specific antigen (PSA) less than 1 ng/ml were integrated to give concentrations of 97 to 178 mM. Semen from a 47-year-old man with benign prostatic hyperplasia and a PSA of 5.5 ng/ml contained 156 mM citrate. In contrast, seminal plasma from 2 patients with prostate cancer, including a 46-year-old man with Gleason grade 8 and PSA 45.2 ng/ml, and a 64-year-old man with grade 6 and PSA 13.0 ng/ml, revealed citrate NMR signals corresponding to a concentration of only 28 and 24 mM, respectively. Spectra from prostatic massage fluid from a normal 23-year-old volunteer showed a citrate of 483 mM, while massage fluid from a 56-year-old patient with Gleason grade 4 cancer showed a citrate of only 1.35 mM. To our knowledge this study is the first to use high resolution NMR of semen to diagnose prostate cancer. Given the known effects of adenocarcinoma on prostate metabolism, the study indicates that high resolution H NMR can be used to measure citrate in seminal fluid, potentially providing a new, rapid, noninvasive screening method.

Metabolic changes in Atlantic salmon exposed to Aeromonas salmonicida detected by 1H-nuclear magnetic resonance spectroscopy of plasma

1H-NMR (nuclear magnetic resonance)-based chemometric methods have been applied for the first time to investigate changes in the plasma metabolite profiles of Atlantic salmon Salmo salar as a result of exposure to Aeromonas salmonicida subsp. salmonicida, a Gram-negative bacterium that is the etiological agent of furunculosis. Plasma samples were obtained from salmon that survived 21 d post exposure to A. salmonicida, and from a control group maintained under similar conditions. 1D 1H-NMR spectra were acquired and principal components analysis (PCA) was used to assess differences between the spectral profiles of plasma from salmon that survived an A. salmonicida challenge, and non-infected controls. PCA enables simultaneous comparison of spectra, presenting a simplified overview of the relationship between spectral data, where spectra cluster based on metabolite profile similarities and differences; information regarding the metabolite variations can therefore be readily deciphered. The major metabolite changes responsible for the spectral differences were related to modification in the lipoprotein profile and choline-based residues, with minor changes in carbohydrates, glycerol, trimethylamine-N-oxide and betaine. These changes indicated that exposure to A. salmonicida induced a characteristic biochemical response which could be used to determine the health status of salmon. This study suggests that with further development this metabolite profiling technique may be a useful tool for diagnosis of disease states in salmon and could provide a better understanding of the host-pathogen relationship which at present is poorly understood for A. salmonicida and Atlantic salmon.

Detection of acute cardiac rejection by high resolution proton magnetic resonance spectroscopy of plasma

Detection of acute cardiac rejection by high resolution proton magnetic resonance spectroscopy of plasma

Evaluation of renal medulla injury after cold preservation and transplantation: noninvasive determination of medullar damage by proton nuclear magnetic resonance spectroscopy of urine and plasma

PRIMARY and early allograft dysfunction remains a challenge for the nephrologist. Usually, the assessment of renal graft dysfunction following transplantation is based on nonspecific measurement of renal function or an invasive method such as transplant biopsy. A rapid diagnosis is necessary because a delay in treating early rejection deteriorates the prognosis of the allograft function. In addition, the antirejection treatment based on a mere clinical diagnosis of rejection could improve delayed graft function caused by acute tubular necrosis or cyclosporine damage through possible side effects of antirejection treatment. In the present study, we wanted to assess whether proton nuclear magnetic resonance (HNMR) spectroscopy can detect ischemic damage and predict acute tubular necrosis after pig kidney preservation in two preservation solutions (Euro-Collins: EC and University of Wisconsin: UW).

Detection of acute cardiac rejection by high resolution proton magnetic resonance spectroscopy of plasma.

We have developed a low-cost and minimally-invasive MRS CDE combined procedure to diagnose routinely low grade rejection, allowing to eliminate systematic biopsies after the first year and to reduce the number of biopsies to the minimum required by the occurrence of immunological and or functional perturbations. The MRS CDE approach is also used to finetune the immunosuppressive treatment.

Nuclear magnetic resonance spectroscopy of plasma to distinguish between malignant and benign diseases causing jaundice and cholestasis.

The sera of 51 patients with malignant (n = 25) and benign (n = 26) hepatopancreatobiliary disorders were analysed by 1H magnetic resonance spectroscopy (NMR) in order to distinguish between malignant and benign diseases causing jaundice and/or cholestasis. Macromolecular linewidths were determined both manually and automatically with a computed analysis, and both methylene (CH2) and methyl (CH3) resonances were evaluated. The mean linewidth of the CH3 peak was significantly narrower in the patients with malignant disease than in the patients with benign disease both in the manual and computed analyses, but no significant differences in the CH2 peak were detected. Diagnostic sensitivity and specificity of the CH3 peak determined in the computed analysis were 92% and 27% respectively. In the light of the current study, it seems obvious that because overlap between benign and malignant groups was too great, 1H NMR spectroscopy of plasma is not of practical value in distinguishing between benign and malignant causes of jaundice and/or cholestasis.

Failure to detect early breast cancer using in vitro nuclear magnetic resonance spectroscopy of plasma.

Water suppressed proton nuclear magnetic resonance (1H NMR) spectroscopy of human plasma has been described as successful in detection of malignancy. We designed a prospective study to test the hypothesis that in vitro NMR spectroscopy has a high sensitivity for detecting early breast cancer. One hundred and thirty-five women were referred for breast biopsy due to abnormal mammograms. One hundred of these were recruited through a population-based mammography screening project. Sixty-nine of 135 women were found to have breast cancer and their average line width of the methyl and methylene resonance in the plasma were compared to those women who had a benign or normal histopathology in the biopsy and to the line width for 100 healthy subjects from the same population. The mean line width at a half-height of the methyl and methylene resonances of the serum lipoprotein lipids in the NMR spectrum did not differ appreciably between the groups. The line width correlated highly with the serum triglycerides, but correction for the level of triglycerides did not improve the diagnostic accuracy of the line width. Receiver-operating characteristic analysis revealed a sensitivity of 61% and a false positive rate of 43% at the most beneficial cut-off of line width (39.7 Hz). In vitro NMR spectroscopy in our hands was thus not a useful diagnostic tool in patients with early breast cancer.

Cephaloridine-induced nephrotoxicity in the fischer 344 rat proton NMR spectroscopic studies of urine and plasma in relation to conventional clinical chemical and histopathological assessments of nephronal damage

The acute toxicological effects of the nephrotoxic antibiotic cephaloridine (CPH, 0–1500 mg/kg) in male Fischer 344 (F344) rats, have been investigated over 48 h using clinical chemistry, histopathology and proton nuclear magnetic resonance (1H NMR) spectroscopy of urine and plasma. High field (400 and 600 MHz)1H NMR urinalysis revealed increased excretion of lactic acid, acetoacetate, alanine, valine, lysine, glutamine and glutamate and a severe, time-dependent glycosuria. A major change observed in urine of CPH-treated animals was the dose-dependent increase in HB which may relate to altered energy metabolism. CPH also caused dose-dependent decreases in the urinary excretion of hippurate, allantoin and protein (conventional assay). This abnormal metabolic profile is consistent with a functional defect in the S1/S2 regions of the proximal tubule, and was confirmed by histologypost mortem. Functional changes observed included elevations in blood urea nitrogen (BUN) and urine flow rate (UFR) and dose-related decreases in urine osmolality. Spin-echo1H NMR spectroscopic analysis of lyophilised plasma, reconstituted with2H2O revealed an abnormal phase modulation of the methyl signal from free alanine and it is postulated that this is due to the release of transaminases from damaged tissue which via a reversible conversion to pyruvate, cause variable deuteration of alanine at the α-CH position. This observation suggests that1H NMR spectral patterns are also dependent on the level of plasma transaminases and this may provide a novel indicator of tissue damage.

Proton Nuclear Magnetic Resonance Spectroscopy of Urine and Plasma in Renal Transplantation Follow-Up

Urine and plasma samples from 39 patients who underwent renal transplantation were analyzed by proton nuclear magnetic resonance (NMR) spectroscopy. The most relevant resonances for evaluating renal function after transplantation were those arising from citrate, trimethylamine-N-oxide (TMAO), alanine, and lactate when compared to creatinine. A resonance at 3.7 ppm was related to cyclosporine toxicity when associated with elevated levels of TMAO. The respective variations in these metabolites in urine could contribute to prognosis and diagnosis of renal function impairment related to cyclosporine toxicity or overdosage, or to rejection. Thus, NMR spectroscopy should improve the follow-up and management of renal transplantation patients.

Cephaloridine-induced nephrotoxicity in the Fischer 344 rat: proton NMR spectroscopic studies of urine and plasma in relation to conventional clinical chemical and histopathological assessments of nephronal damage.

The acute toxicological effects of the nephrotoxic antibiotic cephaloridine (CPH, 0-1500 mg/kg) in male Fischer 344 (F344) rats, have been investigated over 48 h using clinical chemistry, histopathology and proton nuclear magnetic resonance (1H NMR) spectroscopy of urine and plasma. High field (400 and 600 MHz)1H NMR urinalysis revealed increased excretion of lactic acid, acetoacetate, alanine, valine, lysine, glutamine and glutamate and a severe, time-dependent glycosuria. A major change observed in urine of CPH-treated animals was the dose-dependent increase in HB which may relate to altered energy metabolism. CPH also caused dose-dependent decreases in the urinary excretion of hippurate, allantoin and protein (conventional assay). This abnormal metabolic profile is consistent with a functional defect in the S1/S2 regions of the proximal tubule, and was confirmed by histology post mortem. Functional changes observed included elevations in blood urea nitrogen (BUN) and urine flow rate (UFR) and dose-related decreases in urine osmolality. Spin-echo 1H NMR spectroscopic analysis of lyophilised plasma, reconstituted with 2H2O revealed an abnormal phase modulation of the methyl signal from free alanine and it is postulated that this is due to the release of transaminases from damaged tissue which via a reversible conversion to pyruvate, cause variable deuteration of alanine at the alpha-CH position. This observation suggests that 1H NMR spectral patterns are also dependent on the level of plasma transaminases and this may provide a novel indicator of tissue damage.

Use of proton nuclear magnetic resonance spectroscopy of plasma in screening for malignant disease.

In 1986, narrow plasma proton nuclear magnetic resonance (NMR) methyl and methylene line widths were reported to be associated with malignant disease, but more recent studies have not confirmed this relationship. The authors analyzed 106 plasma samples from healthy control subjects and patients with cirrhosis, hepatocellular carcinoma, metastatic liver tumors, other untreated cancers, and hyperlipidemia. NMR spectroscopy was performed using a proton NMR spectrometer operating at 399.65 MHz. A significant difference was found between the mean line widths of the plasma methyl resonances in control subjects and those in patients with cancer or hyperlipidemia. However, no significant difference was found between the mean plasma methylene line widths in control subjects and patients with cancer. Plasma samples from patients with liver disease or hyperlipidemia showed a characteristic methylene spectral pattern. The methylene pattern could be separated into three types: type A had a small peak on the right shoulder of the main peak; type B was a sharp single peak; and type C was a broad single peak. All control subjects had type A pattern; patients with liver disease had type C pattern; and patients who had hyperlipidemia had type B pattern, and hyperlipidemia may affect methyl and methylene line widths in NMR spectra. Because the methyl and methylene levels and their average line widths correlated inversely with triglyceride levels, considering the spectral patterns that indicate hyperlipidemia should decrease false-positive results and make the methyl line width useful for cancer screening.