ABSTRACT:
The use of reliable biomarkers is becoming increasingly important for the improved management of patients with acute and chronic kidney diseases. Recent developments have identified a number of novel biomarkers in serum or urine that can determine the potential risk of kidney damage, distinguish different types of renal injury, predict the progression of disease and have the potential to assess the efficacy of therapeutic intervention. Some of these biomarkers can be used independently while others are more beneficial when used in combination with knowledge of other clinical risk factors. Advances in gene expression analysis, chromatography, mass spectrometry and the development of sensitive enzyme-linked immunosorbent assays have facilitated accurate quantification of many biomarkers. This review primarily focuses on describing new and established biomarkers, which identify and measure the various pathophysiological processes that promote kidney disease. It provides an overview of some of the different classes of renal biomarkers that can be assessed in serum/plasma and urine, including markers of renal function, oxidative stress, structural and cellular injury, immune responses and fibrosis. However, it does not explore the current status of these biomarkers in terms of their clinical validation.
Friday
Thursday
Classical maple syrup urine disease and brain development: Principles of management and formula design.
Branched-chain ketoacid dehydrogenase deficiency results in complex and volatile metabolic derangements that threaten brain development.
Treatment for classical maple syrup urine disease (MSUD) should address this underlying physiology while also protecting children from nutrient deficiencies. Based on a 20-year experience managing 79 patients, we designed a study formula to (1) optimize transport of seven amino acids (Tyr, Trp, His, Met, Thr, Gln, Phe) that compete with branched-chain amino acids (BCAAs) for entry into the brain via a common transporter (LAT1), (2) compensate for episodic depletions of glutamine, glutamate, and alanine caused by reverse transamination, and (3) correct deficiencies of omega-3 essential fatty acids, zinc, and selenium widespread among MSUD patients.
The formula was enriched with LAT1 amino acid substrates, glutamine, alanine, zinc, selenium, and alpha-linolenic acid (18:3n-3). Fifteen Old Order Mennonite children were started on study formula between birth and 34months of age and seen at least monthly in the office. Amino acid levels were checked once weekly and more often during illnesses. All children grew and developed normally over a period of 14-33months. Energy demand, leucine tolerance, and protein accretion were tightly linked during periods of normal growth. Rapid shifts to net protein degradation occurred during illnesses. At baseline, most LAT1 substrates varied inversely with plasma leucine, and their calculated rates of brain uptake were 20-68% below normal. Treatment with study formula increased plasma concentrations of LAT1 substrates and normalized their calculated uptakes into the nervous system. Red cell membrane omega-3 polyunsaturated fatty acids and serum zinc and selenium levels increased on study formula. However, selenium and docosahexaenoic acid (22:6n-3) levels remained below normal. During the study period, hospitalizations decreased from 0.35 to 0.14 per patient per year. There were 28 hospitalizations managed with MSUD hyperalimentation solution; 86% were precipitated by common infections, especially vomiting and gastroenteritis. The large majority of catabolic illnesses were managed successfully at home using 'sick-day' formula and frequent amino acid monitoring. We conclude that the study formula is safe and effective for the treatment of classical MSUD. In principle, dietary enrichment protects the brain against deficiency of amino acids used for protein accretion, neurotransmitter synthesis, and methyl group transfer. Although the pathophysiology of MSUD can be addressed through rational formula design, this does not replace the need for vigilant clinical monitoring, frequent measurement of the complete amino acid profile, and ongoing dietary adjustments that match nutritional intake to the metabolic demands of growth and illness.
Strauss KA, Wardley B, Robinson D, Hendrickson C, Rider NL, Puffenberger EG, Shelmer D, Moser AB, Morton DH.
Clinic for Special Children, Strasburg, PA, USA; Lancaster General Hospital, Lancaster, PA, USA; Department of Biology, Franklin and Marshall College, Lancaster, PA, USA.
Treatment for classical maple syrup urine disease (MSUD) should address this underlying physiology while also protecting children from nutrient deficiencies. Based on a 20-year experience managing 79 patients, we designed a study formula to (1) optimize transport of seven amino acids (Tyr, Trp, His, Met, Thr, Gln, Phe) that compete with branched-chain amino acids (BCAAs) for entry into the brain via a common transporter (LAT1), (2) compensate for episodic depletions of glutamine, glutamate, and alanine caused by reverse transamination, and (3) correct deficiencies of omega-3 essential fatty acids, zinc, and selenium widespread among MSUD patients.
The formula was enriched with LAT1 amino acid substrates, glutamine, alanine, zinc, selenium, and alpha-linolenic acid (18:3n-3). Fifteen Old Order Mennonite children were started on study formula between birth and 34months of age and seen at least monthly in the office. Amino acid levels were checked once weekly and more often during illnesses. All children grew and developed normally over a period of 14-33months. Energy demand, leucine tolerance, and protein accretion were tightly linked during periods of normal growth. Rapid shifts to net protein degradation occurred during illnesses. At baseline, most LAT1 substrates varied inversely with plasma leucine, and their calculated rates of brain uptake were 20-68% below normal. Treatment with study formula increased plasma concentrations of LAT1 substrates and normalized their calculated uptakes into the nervous system. Red cell membrane omega-3 polyunsaturated fatty acids and serum zinc and selenium levels increased on study formula. However, selenium and docosahexaenoic acid (22:6n-3) levels remained below normal. During the study period, hospitalizations decreased from 0.35 to 0.14 per patient per year. There were 28 hospitalizations managed with MSUD hyperalimentation solution; 86% were precipitated by common infections, especially vomiting and gastroenteritis. The large majority of catabolic illnesses were managed successfully at home using 'sick-day' formula and frequent amino acid monitoring. We conclude that the study formula is safe and effective for the treatment of classical MSUD. In principle, dietary enrichment protects the brain against deficiency of amino acids used for protein accretion, neurotransmitter synthesis, and methyl group transfer. Although the pathophysiology of MSUD can be addressed through rational formula design, this does not replace the need for vigilant clinical monitoring, frequent measurement of the complete amino acid profile, and ongoing dietary adjustments that match nutritional intake to the metabolic demands of growth and illness.
Strauss KA, Wardley B, Robinson D, Hendrickson C, Rider NL, Puffenberger EG, Shelmer D, Moser AB, Morton DH.
Clinic for Special Children, Strasburg, PA, USA; Lancaster General Hospital, Lancaster, PA, USA; Department of Biology, Franklin and Marshall College, Lancaster, PA, USA.
Tuesday
Clinical collection and protein properties of expressed prostatic secretions as a source for biomarkers of prostatic disease
Journal of Proteomics
Volume 72, Issue 6, 20 August 2009, Pages 907-917
Abstract
The prostate gland secretes many proteins in a prostatic fluid that combines with seminal vesicle derived fluids to promote sperm activation and function. Proximal fluids of the prostate that can be collected clinically are seminal plasma and expressed-prostatic secretion (EPS) fluids. EPS represents the fluid being secreted by the prostate following a digital rectal prostate massage, which in turn can be collected in voided urine post-exam. This collection is not disruptive to a standard urological exam, and it can be repeatedly collected from men across all prostatic disease states.
A direct EPS fluid can also be collected under anesthesia prior to prostatectomy. While multiple genetic assays for prostate cancer detection are being developed for the shed epithelial cell fraction of EPS urines, the remaining fluid that contains many prostate-derived proteins has been minimally characterized. Approaches to optimization and standardization of EPS collection consistent with current urological exam and surgical practices are described, and initial proteomic and glycomic evaluations of the of EPS fluid are summarized for prostate specific antigen and prostatic acid phosphatase.
Continued characterization of the prostate specific protein components of EPS urine combined with optimization of clinical collection procedures should facilitate discovery of new biomarkers for prostate cancer.
Volume 72, Issue 6, 20 August 2009, Pages 907-917
Abstract
The prostate gland secretes many proteins in a prostatic fluid that combines with seminal vesicle derived fluids to promote sperm activation and function. Proximal fluids of the prostate that can be collected clinically are seminal plasma and expressed-prostatic secretion (EPS) fluids. EPS represents the fluid being secreted by the prostate following a digital rectal prostate massage, which in turn can be collected in voided urine post-exam. This collection is not disruptive to a standard urological exam, and it can be repeatedly collected from men across all prostatic disease states.
A direct EPS fluid can also be collected under anesthesia prior to prostatectomy. While multiple genetic assays for prostate cancer detection are being developed for the shed epithelial cell fraction of EPS urines, the remaining fluid that contains many prostate-derived proteins has been minimally characterized. Approaches to optimization and standardization of EPS collection consistent with current urological exam and surgical practices are described, and initial proteomic and glycomic evaluations of the of EPS fluid are summarized for prostate specific antigen and prostatic acid phosphatase.
Continued characterization of the prostate specific protein components of EPS urine combined with optimization of clinical collection procedures should facilitate discovery of new biomarkers for prostate cancer.
Plasma and urine biomarkers in acute viral hepatitis E
Background
Hepatitis E, caused by the hepatitis E virus (HEV), is endemic to developing countries where it manifests as waterborne outbreaks and sporadic cases. Though generally self-limited with a low mortality rate, some cases progress to fulminant hepatic failure (FHF) with high mortality. With no identified predictive or diagnostic markers, the events leading to disease exacerbation are not known. Our aim is to use proteomic tools to identify biomarkers of acute and fulminant hepatitis E.
Results
We analyzed proteins in the plasma and urine of hepatitis E patients and healthy controls by two-dimensional Differential Imaging Gel Electrophoresis (DIGE) and mass spectrometry, and identified over 30 proteins to be differentially expressed during acute hepatitis E. The levels of one plasma protein, transthyretin, and one urine protein, alpha-1-microglobulin were then quantitated by enzyme immunoassay (EIA) in clinical samples from a larger group of patients and controls. The results showed decreased plasma transthyretin levels (p < 0.005) and increased urine α1m levels (p < 0.001) in acute hepatitis E patients, compared to healthy controls. Preliminary results also showed lower urine zinc alpha glycoprotein levels in fulminant hepatitis E compared to acute disease; this remains to be confirmed with more fulminant cases.
Conclusion
Our results demonstrate the utility of characterizing plasma and urine proteomes for signatures of the host response to HEV infection. We predict that plasma transthyretin and urine alpha-1-microglobulin could be reliable biomarkers of acute hepatitis E. Besides the utility of this approach to biomarker discovery, proteome-level changes in human biofluids would also guide towards a better understanding of host-virus interaction and disease.
Shikha Taneja1 , Somdutta Sen2 , Vijay K Gupta3 , Rakesh Aggarwal4 and Shahid Jameel
1Virology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi - 110067, India
2The Centre for genomic Applications, Okhla Industrial Area (Phase III), New Delhi - 110020, India
3Department of Gastroenterology, Army Hospital, Delhi Cantonment, New Delhi - 110010, IndiaDepartment of Gastroenterology, Sanjay Gandhi Postgraduate Institute for Medical Sciences, Rae Bareilly Road, Lucknow - 226014, India
Hepatitis E, caused by the hepatitis E virus (HEV), is endemic to developing countries where it manifests as waterborne outbreaks and sporadic cases. Though generally self-limited with a low mortality rate, some cases progress to fulminant hepatic failure (FHF) with high mortality. With no identified predictive or diagnostic markers, the events leading to disease exacerbation are not known. Our aim is to use proteomic tools to identify biomarkers of acute and fulminant hepatitis E.
Results
We analyzed proteins in the plasma and urine of hepatitis E patients and healthy controls by two-dimensional Differential Imaging Gel Electrophoresis (DIGE) and mass spectrometry, and identified over 30 proteins to be differentially expressed during acute hepatitis E. The levels of one plasma protein, transthyretin, and one urine protein, alpha-1-microglobulin were then quantitated by enzyme immunoassay (EIA) in clinical samples from a larger group of patients and controls. The results showed decreased plasma transthyretin levels (p < 0.005) and increased urine α1m levels (p < 0.001) in acute hepatitis E patients, compared to healthy controls. Preliminary results also showed lower urine zinc alpha glycoprotein levels in fulminant hepatitis E compared to acute disease; this remains to be confirmed with more fulminant cases.
Conclusion
Our results demonstrate the utility of characterizing plasma and urine proteomes for signatures of the host response to HEV infection. We predict that plasma transthyretin and urine alpha-1-microglobulin could be reliable biomarkers of acute hepatitis E. Besides the utility of this approach to biomarker discovery, proteome-level changes in human biofluids would also guide towards a better understanding of host-virus interaction and disease.
Shikha Taneja1 , Somdutta Sen2 , Vijay K Gupta3 , Rakesh Aggarwal4 and Shahid Jameel
1Virology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi - 110067, India
2The Centre for genomic Applications, Okhla Industrial Area (Phase III), New Delhi - 110020, India
3Department of Gastroenterology, Army Hospital, Delhi Cantonment, New Delhi - 110010, IndiaDepartment of Gastroenterology, Sanjay Gandhi Postgraduate Institute for Medical Sciences, Rae Bareilly Road, Lucknow - 226014, India
Thursday
Three new Cardiovascular Risk Markers - Do They Improve Risk Prediction and Influence Treatment
Olsen MH, Sehestedt T, Lyngbæk S, Hansen TW, Rasmussen S, Wachtell K, Torp-Pedersen C, Hildebrandt PR, Ibsen H.
Research Center for Prevention and Health, Gentofte University Hospital, Holbaek Hospital, Denmark
In order to prioritize limited health resources in a time of increasing demands optimal cardiovascular risk stratification is essential. We tested the additive prognostic value of 3 relatively new, but established cardiovascular risk markers: N-terminal pro brain natriuretic peptide (Nt-proBNP), related to hemodynamic cardiovascular risk factors, high sensitivity C-reactive protein (hsCRP), related to metabolic cardiovascular risk factors and urine albumin/creatinine ratio (UACR), related to hemodynamic as well as metabolic risk factors.
In healthy subjects with a 10-year risk of cardiovascular death lower than 5% based on HeartScore and therefore not eligible for primary prevention, the actual 10-year risk of cardiovascular death exceeded 5% in a small subgroup of subjects with UACR higher than the 95-percentile of approximately 1.6 mg/mmol. C
ombined use of high UACR or high hsCRP identified a larger subgroup of 16% with high cardiovascular risk in which primary prevention may be advised despite low-moderate cardiovascular risk based on HeartScore. Furthermore, combined use of high UACR or high Nt-proBNP in subjects with known cardiovascular disease or diabetes identified a large subgroup of 48% with extremely high cardiovascular risk who should be referred for specialist care to optimize treatment
Research Center for Prevention and Health, Gentofte University Hospital, Holbaek Hospital, Denmark
In order to prioritize limited health resources in a time of increasing demands optimal cardiovascular risk stratification is essential. We tested the additive prognostic value of 3 relatively new, but established cardiovascular risk markers: N-terminal pro brain natriuretic peptide (Nt-proBNP), related to hemodynamic cardiovascular risk factors, high sensitivity C-reactive protein (hsCRP), related to metabolic cardiovascular risk factors and urine albumin/creatinine ratio (UACR), related to hemodynamic as well as metabolic risk factors.
In healthy subjects with a 10-year risk of cardiovascular death lower than 5% based on HeartScore and therefore not eligible for primary prevention, the actual 10-year risk of cardiovascular death exceeded 5% in a small subgroup of subjects with UACR higher than the 95-percentile of approximately 1.6 mg/mmol. C
ombined use of high UACR or high hsCRP identified a larger subgroup of 16% with high cardiovascular risk in which primary prevention may be advised despite low-moderate cardiovascular risk based on HeartScore. Furthermore, combined use of high UACR or high Nt-proBNP in subjects with known cardiovascular disease or diabetes identified a large subgroup of 48% with extremely high cardiovascular risk who should be referred for specialist care to optimize treatment
Tuesday
Urine Test May Identify Aggressive Prostate Cancer
An experimental urine test is "at least as good" as the prostate specific antigen (PSA) test for predicting which men have aggressive prostate cancer, according to a University of Michigan researcher who participated in a new study published online in the February 12 issue of Nature.
The urine test, which assesses levels of prostate-cancer-specific metabolites, could eventually be added to PSA and other tools for monitoring prostate cancer progression, said study coauthor John Wei, MD, at a press briefing. Dr. Wei is an associate professor of urology at the University of Michigan Medical School, in Ann Arbor.
However, the new study had a small sample, and the scientific approach to analyze metabolites as prostate cancer biomarkers needs further validation and development, he added.
Nonetheless, Dr. Wei and his colleagues believe that 1 of the metabolites, sarcosine, has the potential to differentiate between benign prostate tissue and localized/metastatic prostate cancer.
"One of the main clinical issues in prostate cancer is distinguishing between aggressive [and] indolent slow-growing disease," said senior author Arul Chinnaiyan, MD, PhD, during the press briefing. The inability to do so leads to "overtreatment," added Dr. Chinnaiyan, who is director of the Michigan Center for Translational Pathology, in Ann Arbor.
In the new study, the researchers identified at least 10 prostate-cancer-specific metabolites. Notably, sarcosine was increased "most robustly in metastatic prostate cancer," in comparison with the others, they write. It was also elevated in locally advanced prostate cancer, but less so, added Dr. Chinnaiyan.
Dr. Chinnaiyan's lab has also developed urine tests to screen for prostate cancer that are more accurate than PSA tests, as reported by Medscape Oncology. But the tests employ genetic biomarkers and are for screening, not monitoring, the disease.
The new study is the first time that "metabolomics," which surveys the metabolite composition of cells and tissues, akin to the way genetics surveys their genetic composition, has been shown "to solve a real-world problem," said the researchers at the press briefing.
Why Prostate-Cancer-Specific Metabolites?
In the study, the investigators examined 1126 metabolites across 262 samples of tissue, blood, or urine associated with benign prostate tissue, early-stage prostate cancer, and metastatic prostate cancer.
Of the metabolites that were present more often in prostate cancer than in the benign cells, sarcosine appeared to be the strongest indicator of advanced disease. Levels of sarcosine, an amino acid, were elevated in 79% of the metastatic prostate cancer samples and in 42% of the locally advanced cancer samples. Sarcosine was not found in the cancer-free samples.
In the study, sarcosine was a better indicator of advancing aggressive disease than the PSA test, which both monitors and screens for prostate cancer.
"When we're looking at metabolites, we're looking several steps beyond genes and proteins. It allows us to look very deeply at some of the functions of the cells and the biochemistry that occurs during cancer development," added Dr. Chinnaiyan in a statement.
The researchers also showed that adding sarcosine to cultures of benign prostate cells turned them into invasive cancer cells, suggesting that the molecule may have an important role in disease.
"Components of the sarcosine pathway may have potential as biomarkers of prostate cancer prevention and serve as new avenues for therapeutic intervention," note the authors.
The Future of Prostate Cancer Testing
"Current biomarkers for detection or progression of prostate cancer are not as precise as we would like. Therefore, a more accurate indicator of cancer is of great interest," said Sudhir Srivastava, PhD, chief of the National Cancer Institute (NCI)'s Cancer Biomarkers Research Group, in a statement. "Sarcosine and some other select metabolites may be excellent indicators of cancer progression."
The NCI Early Detection Research Network supported the study, but the Michigan researchers emphasized that their urine test is not a screening test. "We only examined sarcosine in the context of aggressive prostate cancer," noted Dr. Wei. "A lot more work has to be done before it can be used as a screening test."
Sarcosine was detected in the urine, which makes researchers hopeful that a simple urine test could be used to monitor the disease.
Dr. Wei imagined that the combination of the PSA test and new biomarkers, such as prostate-cancer-specific metabolites (including sarcosine and some of the others identified in the new study), will allow clinicians to eventually "individualize" the analysis of patients. "This may allow clinicians to determine — before biopsy — the severity of disease," he speculated.
The study was funded by the National Cancer Institute Early Detection Research Network, National Institutes of Health, an MTTC grant, the Burroughs Welcome Foundation, and the Doris Duke Charitable Foundation.
Nature. 2009.457:910-915, 799-800.
The urine test, which assesses levels of prostate-cancer-specific metabolites, could eventually be added to PSA and other tools for monitoring prostate cancer progression, said study coauthor John Wei, MD, at a press briefing. Dr. Wei is an associate professor of urology at the University of Michigan Medical School, in Ann Arbor.
However, the new study had a small sample, and the scientific approach to analyze metabolites as prostate cancer biomarkers needs further validation and development, he added.
Nonetheless, Dr. Wei and his colleagues believe that 1 of the metabolites, sarcosine, has the potential to differentiate between benign prostate tissue and localized/metastatic prostate cancer.
"One of the main clinical issues in prostate cancer is distinguishing between aggressive [and] indolent slow-growing disease," said senior author Arul Chinnaiyan, MD, PhD, during the press briefing. The inability to do so leads to "overtreatment," added Dr. Chinnaiyan, who is director of the Michigan Center for Translational Pathology, in Ann Arbor.
In the new study, the researchers identified at least 10 prostate-cancer-specific metabolites. Notably, sarcosine was increased "most robustly in metastatic prostate cancer," in comparison with the others, they write. It was also elevated in locally advanced prostate cancer, but less so, added Dr. Chinnaiyan.
Dr. Chinnaiyan's lab has also developed urine tests to screen for prostate cancer that are more accurate than PSA tests, as reported by Medscape Oncology. But the tests employ genetic biomarkers and are for screening, not monitoring, the disease.
The new study is the first time that "metabolomics," which surveys the metabolite composition of cells and tissues, akin to the way genetics surveys their genetic composition, has been shown "to solve a real-world problem," said the researchers at the press briefing.
Why Prostate-Cancer-Specific Metabolites?
In the study, the investigators examined 1126 metabolites across 262 samples of tissue, blood, or urine associated with benign prostate tissue, early-stage prostate cancer, and metastatic prostate cancer.
Of the metabolites that were present more often in prostate cancer than in the benign cells, sarcosine appeared to be the strongest indicator of advanced disease. Levels of sarcosine, an amino acid, were elevated in 79% of the metastatic prostate cancer samples and in 42% of the locally advanced cancer samples. Sarcosine was not found in the cancer-free samples.
In the study, sarcosine was a better indicator of advancing aggressive disease than the PSA test, which both monitors and screens for prostate cancer.
"When we're looking at metabolites, we're looking several steps beyond genes and proteins. It allows us to look very deeply at some of the functions of the cells and the biochemistry that occurs during cancer development," added Dr. Chinnaiyan in a statement.
The researchers also showed that adding sarcosine to cultures of benign prostate cells turned them into invasive cancer cells, suggesting that the molecule may have an important role in disease.
"Components of the sarcosine pathway may have potential as biomarkers of prostate cancer prevention and serve as new avenues for therapeutic intervention," note the authors.
The Future of Prostate Cancer Testing
"Current biomarkers for detection or progression of prostate cancer are not as precise as we would like. Therefore, a more accurate indicator of cancer is of great interest," said Sudhir Srivastava, PhD, chief of the National Cancer Institute (NCI)'s Cancer Biomarkers Research Group, in a statement. "Sarcosine and some other select metabolites may be excellent indicators of cancer progression."
The NCI Early Detection Research Network supported the study, but the Michigan researchers emphasized that their urine test is not a screening test. "We only examined sarcosine in the context of aggressive prostate cancer," noted Dr. Wei. "A lot more work has to be done before it can be used as a screening test."
Sarcosine was detected in the urine, which makes researchers hopeful that a simple urine test could be used to monitor the disease.
Dr. Wei imagined that the combination of the PSA test and new biomarkers, such as prostate-cancer-specific metabolites (including sarcosine and some of the others identified in the new study), will allow clinicians to eventually "individualize" the analysis of patients. "This may allow clinicians to determine — before biopsy — the severity of disease," he speculated.
The study was funded by the National Cancer Institute Early Detection Research Network, National Institutes of Health, an MTTC grant, the Burroughs Welcome Foundation, and the Doris Duke Charitable Foundation.
Nature. 2009.457:910-915, 799-800.
Global analysis of metabolites in rat and human urine based on gas chromatography/time-of-flight mass spectrometry
Sediment in urine may contain low-molecular-weight compounds that should be included in the analysis. To date, no systematic investigation has addressed this issue. We investigated three primary factors that influence the extraction efficiency of metabolites during preparation of urine samples for metabolomic research: centrifugation, pH, and extraction solvents. Obtained with the use of gas chromatography/time-of-flight mass spectrometry (GC/TOFMS) technique and principal component analysis (PCA), our results indicate that (1) conventional centrifugation causes an apparent loss of some metabolites, indicating that urine samples for metabolomic research should not be centrifuged before procedures are undertaken to recover the metabolites; (2) pH adjustment has a large impact on the recovery of metabolites and is therefore not encouraged; (3) with design of experiment analysis, methanol and water yield the optimal extraction efficiency. Differences between rat urine and human urine were observed and are discussed. Ninety-nine metabolites identified in rat and human urine are presented. An efficient protocol is proposed for the pretreatment of urine samples.
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