The cellular origin and proliferative status of regenerating renal parenchyma after mercuric chloride damage and erythropoietin treatment

OBJECTIVES: In this study, we have sought to establish the cellular origin and proliferative status of the renal parenchyma as it regenerates after damage induced by mercuric chloride, with or without erythropoietin treatments, that might alter the response. MATERIALS AND METHODS: Female mice were irradiated and male whole bone marrow was transplanted into them. Six weeks later recipient mice were assigned to one of four groups: control, mercuric chloride treated, erythropoietin treated and treated with mercuric chloride plus erythropoietin. RESULTS: Tubular injury scores were high 3 days after mercuric chloride and had recovered partially after 14 days, in line with serum urea nitrogen levels. Confocal microscopy confirmed the tubular location of bone marrow-derived cells. A 'four-in-one' analytical technique (identifying cell origin, tubular phenotype, tubular basement membranes and S-phase status) revealed that tubular necrosis increased bone marrow derivation of renal tubular epithelium from a baseline of approximately 1.3% to approximately 4.0%. Erythropoietin increased the haematocrit, but no other effects were detected. CONCLUSION: As 1 in 12 proximal tubular cells in S-phase was derived from bone marrow, we conclude that in the kidney, the presence of bone marrow-derived cells makes a minor but important regenerative contribution after tubular necrosis.     

Mercuric chloride-induced nephrotoxicity in the rat following unilateral nephrectomy and compensatory renal growth

The nephropathy induced by mercuric chloride was assessed in unilaterally nephrectomized (NPX) and sham-operated (SO) rats using histological and urinalysis techniques. This assessment was carried out in order to test whether or not rats are more susceptible to the nephrotoxic effects of mercuric chloride after unilateral nephrectomy and a period allowing for compensatory renal growth. Twelve days after surgery both NPX and SO rats were given a single 1.5, 2.0 or 2.5 mumol/kg dose of mercuric chloride (i.v.). Twenty-four hours after the 1.5 or 2.0 mumol/kg dose of mercuric chloride was administered, cellular and tubular necrosis in the pars recta segments of proximal tubules in the outer medulla was more severe in NPX rats than in SO rats. Moreover, the urinary excretion of a number of cellular enzymes (e.g. lactate dehydrogenase) and plasma solutes (e.g. albumin) was greater in NPX rats than in SO rats. At the 2.5 mumol/kg dose of mercuric chloride, renal tubular damage was quite extensive in both groups of rats; to such an extent that possible differences in renal tubular damage between the NPX and SO rats could not be determined histologically. However, the urinary excretion of alanine aminopeptidase was greater in the NPX rats than in the SO rats. Therefore, based on the aforementioned findings, rats that have undergone and adapted to a reduction in renal mass (i.e. unilateral nephrectomy) appear to be more vulnerable to the nephrotoxic effects of mercuric chloride than rats with two normal kidneys.     

Rat-urine glycosidases and kidney damage

1. The activities of beta-galactosidase, beta-glucosidase, beta-glucuronidase and N-acetyl, beta-glucosaminidase were estimated in normal and pathological rat urine, with 4-methylumbelliferyl glycosides as substrates. 2. Kidney damage induced by injections of uranium nitrate, mercuric chloride, potassium dichromate or 4-nitrophenylarsonic acid causes a marked increase in the urinary excretion of all four enzymes. 3. The rise in beta-glucosidase activity was associated with the appearance of a new urinary enzyme species, which was examined by starch-gel electrophoresis, DEAE-cellulose chromatography and filtration on Sephadex G-75 and G-200. 4. This enzyme appears to be identical with its counterpart in the kidney, and it is suggested that it arises in the urine as a result of renal tubular breakdown. 5. The other glycosidases examined also show some physical similarities to the corresponding enzymes of the rat kidney.     

Urinary enzyme excretion in acute and subacute experimental lithium administration

Blood lithium (Li) levels, renal functional parameters and urine excretion of enzymatic activities having different intracellular sites were investigated on rats submitted to acute and subacute Li chloride administration. In acute experiments increased levels of all detected enzymes were assayed following Li single doses of 5 and 10 mEq/kg b.w. In subacute poisoning, urine output of lactate dehydrogenase, aspartate transaminase and alanine transaminase was significantly over the basal ranges following 15 days in concomitance with marked elevation of plasma Li levels and exhibited progressive increase until 30 days; on the 10th day following Li withdrawal, elevated excretion of enzymatic activities was still assayed. The results are in agreement with data about the localization of the histologic lesions involving different sites of the nephron in acute Li poisoning and the distal tubular tract in subacute toxicity. In subacute administration the output of cytoplasmic and mitochondrial activities can be assumed as an index of damage of the nephron cells which can persist following Li withdrawal. Our findings indicate that the urine enzyme assay is a valuable tool to detect renal damage in experimental Li nephropathy.     

Circulating TNF Receptors 1 and 2 Are Associated with the Severity of Renal Interstitial Fibrosis in IgA Nephropathy

The current study aimed to examine whether the levels of TNF receptors 1 and 2 (TNFR1 and TNFR2) in serum and urine were associated with other markers of kidney injury and renal histological findings, including TNFR expression, in IgA nephropathy (IgAN). The levels of the parameters of interest were measured by immunoassay in 106 biopsy-proven IgAN patients using samples obtained immediately before renal biopsy and in 34 healthy subjects. Renal histological findings were evaluated using immunohistochemistry. The levels of serum TNFRs were higher in IgAN patients than in healthy subjects. The levels of both TNFRs in serum or urine were strongly correlated with each other (r > 0.9). Serum TNFR levels were positively correlated with the urinary protein to creatinine ratio (UPCR) and four markers of tubular damage of interest (N-acetyl-β-D-glucosaminidase [NAG], β2 microglobulin [β2m], liver-type fatty acid-binding protein [L-FABP], and kidney injury molecule-1 [KIM-1]) and negatively correlated with estimated glomerular filtration rate (eGFR). Patients in the highest tertile of serum TNFR levels showed more severe renal interstitial fibrosis than did those in the lowest or second tertiles. The tubulointerstitial TNFR2-, but not TNFR1-, positive area was significantly correlated with the serum levels of TNFRs and eGFR. Stepwise multiple regression analysis revealed that elevated serum TNFR1 or TNFR2 levels were a significant determinant of renal interstitial fibrosis after adjusting for eGFR, UPCR, and other markers of tubular damage. In conclusion, elevated serum TNFR levels were significantly associated with the severity of renal interstitial fibrosis in IgAN patients. However, the source of TNFRs in serum and urine remains unclear.     

Effect of Bacopa monniera on liver and kidney toxicity in chronic use of opioids

In the present study, we investigated the protective effect of Bacopa monniera, an indigenous Ayurvedic medicinal plant in India, against morphine-induced liver and kidney toxicity in rats. Morphine intoxicated rats received 10-160 mg/kg body weight of morphine hydrochloride intraperitoneally for 21 days. Bacopa monniera Extract (BME) pretreated rats were administered with BME (40 mg/kg) orally once a day 2 h before the injection of morphine for 21 days. Pretreatment with BME has shown to possess a significant protective effect against morphine-induced liver and kidney functions in terms of serum glutamate oxaloacetate transaminase, serum glutamate pyruvate transaminase, alkaline phosphatase, lactate dehydrogenases and gamma-glutamyl transferase activities and urea, creatinine and uric acid level respectively. Histopathological changes of liver and kidney were also in accordance with the biochemical findings. The results of this study indicate that Bacopa monniera extract exerted a protection against morphine-induced liver and kidney toxicity.     

Epidermal growth factor attenuates tubular necrosis following mercuric chloride damage by regeneration of indigenous,not bone marrow‐derived cells

To assess effects of epidermal growth factor (EGF) and pegylated granulocyte colony‐stimulating factor (P‐GCSF; pegfilgrastim) administration on the cellular origin of renal tubular epithelium regenerating after acute kidney injury initiated by mercuric chloride (HgCl₂). Female mice were irradiated and male whole bone marrow (BM) was transplanted into them. Six weeks later recipient mice were assigned to one of eight groups: control, P‐GCSF+, EGF+, P‐GCSF+EGF+, HgCl₂, HgCl₂+P‐GCSF+, HgCl₂+EGF+ and HgCl₂+P‐GCSF+EGF+. Following HgCl₂, injection tubular injury scores increased and serum urea nitrogen levels reached uraemia after 3 days, but EGF‐treated groups were resistant to this acute kidney injury. A four‐in‐one analytical technique for identification of cellular origin, tubular phenotype, basement membrane and S‐phase status revealed that BM contributed 1% of proximal tubular epithelium in undamaged kidneys and 3% after HgCl₂ damage, with no effects of exogenous EGF or P‐GCSF. Only 0.5% proximal tubular cells were seen in S‐phase in the undamaged group kidneys; this increased to 7–8% after HgCl₂ damage and to 15% after addition of EGF. Most of the regenerating tubular epithelium originated from the indigenous pool. BM contributed up to 6.6% of the proximal tubular cells in S‐phase after HgCl₂ damage, but only to 3.3% after additional EGF. EGF administration attenuated tubular necrosis following HgCl₂ damage, and the major cause of this protective effect was division of indigenous cells, whereas BM‐derived cells were less responsive. P‐GCSF did not influence damage or regeneration.     

Silibinin ameliorates arsenic induced nephrotoxicity by abrogation of oxidative stress, inflammation and apoptosis in rats

Arsenic (As) is an environmental and industrial pollutant that affects various organs in human and experimental animals. Silibinin is a naturally occurring plant bioflavonoid found in the milk thistle of Silybum marianum, which has been reported to have a wide range of pharmacological properties. A body of evidence has accumulated implicating the free radical generation with subsequent oxidative stress in the biochemical and molecular mechanisms of As toxicity. Since kidney is the critical target organ of chronic As toxicity, we carried out this study to investigate the effects of silibinin on As-induced toxicity in the kidney of rats. In experimental rats, oral administration of sodium arsenite [NaAsO₂, 5?mg/(kg?day)] for 4?weeks significantly induced renal damage which was evident from the increased levels of serum urea, uric acid, creatinine with a significant (p?<?0.05) decrease in creatinine clearance. As also significantly decreased the levels of urea, uric acid and creatinine in urine. A markedly increased levels of lipid peroxidation markers (thiobarbituric acid reactive substances and lipid hydroperoxides) and protein carbonyl contents with significant (p?<?0.05) decrease in non-enzymatic antioxidants (total sulfhydryl groups, reduced glutathione, vitamin C and vitamin E) and enzymatic antioxidants (superoxide dismutase, catalase, glutathione peroxidase and glutathione S-transferase), Glutathione metabolizing enzymes (glutathione reductase and glutathione-6-phosphate dehydrogenase) and membrane bound ATPases were also observed in As treated rats. Co-administration of silibinin (75?mg/kg?day) along with As resulted in a reversal of As-induced biochemical changes in kidney accompanied by a significant decrease in lipid peroxidation and an increase in the level of renal antioxidant defense system. The histopathological and immunohistochemical studies in the kidney of rats also shows that silibinin (75?mg/kg?day) markedly reduced the toxicity of As and preserved the normal histological architecture of the renal tissue, inhibited the caspase-3 mediated tubular cell apoptosis and decreased the NADPH oxidase, iNOS and NF-κB over expression by As and upregulated the Nrf2 expression in the renal tissue. The present study suggests that the nephroprotective potential of silibinin in As toxicity might be due to its antioxidant and metal chelating properties, which could be useful for achieving optimum effects in As-induced renal damage.     

Protection by selenium against gentamicin-induced acute renal damage in the rat

Gentamicin has been shown to induce renal tubular damage in man and laboratory animals and to result in elevated urinary excretion of some enzymes associated with specific cell regions in the kidney. In the present investigation, the possible protective effect of selenium against gentamicin-induced renal damage was tested by measuring the urinary excretion of some enzymes in the presence and absence of selenium. Our results show that a prior subcutaneous injection of selenium to rats for two days followed by a simultaneous S.C. injection of gentamicin and selenium resulted in a marked reduction in the excretion of such biochemical systems as the urine volume, urinary proteins, alkaline and acid phosphatases, beta-glucuronidase, muramidase, and glutamate dehydrogenase. Renal functional studies revealed that selenium-treated rats suffered less adverse effects compared to rats treated with gentamicin alone. Urinary acid phosphatase, beta-glucuronidase and muramidase, the three lysosomal enzymes tested, appeared to respond most readily to protection by selenium.     

Nephroprotective effect of pigmented violacein isolated from Chromobacterium violaceum in wistar rats

The present study aimed to analyze the nephroprotective property of violacein obtained from the bacterium, Chromobacterium violaceum. The nephrotoxicity in the animal model was induced by gentamicin, potassium dichromate, mercuric chloride, and cadmium chloride-induced nephrotoxicity in the Wistar rats was analyzed by measuring the serum creatinine, uric acid, and urea level. The present investigation revealed the nephroprotective property on convoluted proximal tubule (S1 and S2 segments) and the straight proximal tubule (S3 segment). Also, violacein significantly improved the renal function by the renal protective property on S2 segment of proximal tubule from the nephrotoxicity stimulated by mercuric chloride, potassium dichromate, cadmium chloride and gentamicin in animal models. Animal model studies revealed that violacein at 20 and 40 mg/kg p.o improved the renal function and significantly reduced the increased amount of uric acid, creatinine, and blood urea compared to the control.     

A γ-aminobutyrate pathway in mammalian kidney cortex

Rat kidney cortex converts l-glutamate to γ-aminobutyrate by a decarboxylation reaction which differs from the corresponding reaction in brain. Renal l-glutamate decarboxylase has two apparent K_m values for glutamate in homogenates (0.4 and 2.5 mM). γ-Aminobutyrate is converted by a transaminase whose capacity appears to exceed the decarboxylase. γ-Aminobutyrate is converted ultimately to succinate and CO₂.γ-Aminobutyrate stimulates respiration of kidney cortex slices in vitro and the compound crosses cell membranes in kidney by a respiration-linked, mediated process.Chronic acidosis lowers renal γ-aminobutyrate in the rat; brain γ-aminobutyrate is unaffected by acidosis. Glutamic acid decarboxylase and γ-aminobutyrate transaminase activities are unchanged in acidosis. α-Methylglutamate, an inhibitor of renal glutaminase, lowers the γ-aminobutyrate and glutamate content of rat kidney in normal and acidotic states. Aminooxyacetic acid in vivo, an inhibitor of γ-aminobutyrate transaminase, causes a striking increase in renal γ-aminobutyrate during chronic acidosis.At concentrations of glutamate in vitro, which are similar to the tissue glutamate content in vivo, the γ-aminobutyrate pathway accounts for approximately one-fourth of glutamate disposal in rat kidney cortex slices.     

Urinary enzyme excretion and renal lactate dehydrogenase isoenzyme pattern in acute HgCl2 nephropathy of rat

The activity of several enzymes with different intracellular sites was determined in urine at various times following nonfatal acute tubular necrosis induced by mercuric chloride administration. The excretion rate of all tested enzymes rose on the 1st and 2nd day; in the next observations (days 7-15) enzymatic values approached the basal values. The lactate dehydrogenase isoenzyme pattern of the renal cortical zone showed an early shift towards cathodic fractions and later (7 days) an increase of middle ones; the normal anodic zymogram recovered after a suitable time interval (30 days). The isoenzymatic changes are related both to the renal hypoxia and to the appearance of less differentiated cells. The behaviour of functional parameters (urine flow, osmolality, urea clearance, creatinine clearance) were well in agreement with the observed enzyme and renal isoenzyme changes.     

Low Temperature Effects on Soybean (Glycine max [L.] Merr. cv. Wells) Free Amino Acid Pools during Germination

The free amino acid concentrations in cotyledons and axes of soybean (Glycine max [L.] Merr. cv. Wells) seedlings were determined by automated single column analysis after germination at 10 and 23 C. After 5 days germination at 10 C, glutamate and aspartate were in high concentration in both cotyledons and axes (38 and 24% of total free amino acids recovered, respectively), whereas the concentrations of their amide derivatives, asparagine and glutamine, were low in cotyledons (4.4%) and high in axes (21%). In contrast, after 5 days germination at 23 C, asparagine and glutamine accounted for 22 and 45% of total free amino acids in cotyledons and axes respectively, and aspartate and glutamate concentrations were low. The activities of glutamine synthetase and asparagine synthetase were considerably lower in tissues from the 10 C treatment than those from the 23 C treatment.

Aspartate and glutamate concentrations were nearly equal in all but one sample. Both glutamate oxaloacetate transaminase and glutamate dehydrogenase activities were much higher in axis tissues at 23 C as compared to 10 C. Arrhenius plots of axis glutamate oxaloacetate transaminase and glutamate dehydrogenase activities were biphasic and triphasic, respectively, with energies of activation for both increasing with low temperature. Energies of activation were identical for glutamate oxaloacetate transaminase from 10 and 23 C treatments but much higher for glutamate dehydrogenase from 23 C-treated axes. This indicates a difference in enzyme complement for glutamate dehydrogenase with the two treatments.

Hydrolysis of free amino acid sample (basic fraction) aliquots showed large quantities of peptides in 23 C-treated axes at 2 days, while few or no peptides were found in the 10 C treatment. Amino acid residues most prevalent in peptides were aspartate, threonine, serine, glutamate, and glycine.

     

A continuous automated assay of lipolysis during perifusion of isolated fat cells

Cysteine sulfinate transaminase (E.C. 2.6.1,l-cysteine sulfinate:2 oxoglutarate aminotransferase) catalyzes the conversion of cysteine sulfinate and α-ketoglutarate to 3-sulfonyl pyruvate and glutamate. A simple two-step assay has been developed to measure the enzyme activity in the high speed supernatant of whole brain homogenate. In the first step, the supernatant is incubated in the presence of exogenous substrate, then glutamate dehydrogenase is added to catalyze the conversion of glutamate to α-ketoglutarate, and the concomitant production of NADH is fluorimetrically monitored. The apparent K_m values of cysteine sulfinate transaminase for cysteine sulfinate and α-ketoglutarate are 1.24 and 0.22 mm, respectively. This assay is extremely rapid and has a high sensitivity, samples containing as low as 30 ng of protein may be accurately assayed.     

The resting metabolism of dodder seeds

Extracts of mature seeds of Cuscuta reflexa were examined for any deficiency in key enzymes. The activities of malate dehydrogenase, β-amylase and fructose 1,6-diphosphate aldolase exceeded 5.0 μmol substrate/min/g, while those of starch phosphorylase, α-amylase, acid phosphatase, phosphogluconate dehydrogenase (decarboxylating), aspartate aminotransferase, glucose 6-phosphate dehydrogenase, fructose 1,6-diphosphatase and alanine aminotransferase fell within the range 1 to 5 μmol/min/g and hexokinase, isocitrate dehydrogenase and alkaline phosphatase were below 1 μmol substrate/min/g seed powder. No activity of the following were found: acid invertase, alkaline invertase, phytase and glutamate dehydrogenase. Some of these observations were made also for seeds of Cuscuta campestris and Cuscuta indicora.     

Glutamate Biosynthesis in Lactococcus lactis subsp. lactis NCDO 2118

Unlike other lactic acid bacteria, Lactococcus lactis subsp. lactis NCDO 2118 was able to grow in a medium lacking glutamate and the amino acids of the glutamate family. Growth in such a medium proceeded after a lag phase of about 2 days and with a reduced growth rate (0.11 h⁻¹) compared to that in the reference medium containing glutamate (0.16 h⁻¹). The enzymatic studies showed that a phosphoenolpyruvate carboxylase activity was present, while the malic enzyme and the enzymes of the glyoxylic shunt were not detected. As in most anaerobic bacteria, no α-ketoglutarate dehydrogenase activity could be detected, and the citric acid cycle was restricted to a reductive pathway leading to succinate formation and an oxidative branch enabling the synthesis of α-ketoglutarate. The metabolic bottleneck responsible for the limited growth rate was located in this latter pathway. As regards the synthesis of glutamate from α-ketoglutarate, no glutamate dehydrogenase was detected. While the glutamate synthase-glutamine synthetase system was detected at a low level, high transaminase activity was measured. The conversion of α-ketoglutarate to glutamate by the transaminase, the reverse of the normal physiological direction, operated with different amino acids as nitrogen donor. All of the enzymes assayed were shown to be constitutive.     

A new biologically active triterpenoid saponin from the aerial parts of Neanotis wightiana

Phytochemical investigation of the aerial parts of Neanotis wightiana for the first time has led to the isolation of one new triterpenoid saponin, neanoside A (1), along with seven known compounds, oleanolic acid (2), ursolic acid (3), β-sitosterol (4) and its glucoside (5), stigmasterol (6) and its glucoside (7) and hexacosanoic acid (8). The structures of these compounds were elucidated by means of spectroscopic (NMR, MS and other) and chemical techniques as well as comparison with literature data. The structure of 1 was elucidated as 3-O-α-l-rhamnopyranosyl-(1 → 2)-β-d-xylopyranosyl (1 → 3)-β-d-glucopyranosyl bayogenin. The in vitro biochemical analysis of compound 1 against the activity of human serum liposomal enzymes, SGOT (serum glutamate oxaloacetate transaminase), SGPT (serum glutamate pyruvate transaminase) and ALP (alkaline phosphatase) and glycerol kinase showed significant reduction of their activity.     

Properties of γ-aminobutyric acid synthesis by rat renal cortex

Substantial synthesis of γ-aminobutyric acid occurs in rat renal cortex. Renal glutamate decarboxylase activity (24.3±2.9 (S.E.) nmols/mg protein per h) is 15% of that in brain; renal γ-aminobutyric acid content (39.5±5.3 (S.E.) nmols/g wet wt.) is 5% of the whole brain concentration. Properties of glutamate decarboxylase were studied in homogenates of rat renal cortex and rat brain under conditions for which γ-aminobutyric acid formation from [2,3-³H]glutamate and CO₂ release from [1-¹⁴C]glutamate were equal. Several properties of renal glutamate decarboxylase distinguish it from the corresponding brain enzyme: (1) renal glutamate decarboxylase is selectively inhibited by cysteine sulfinic acid (K_i = 5·10^?5 M) ; (20 renal glutamate decarboxylase is less sensitive (K_i = 3–5·10^?5 M)_to inhibition by aminooxyacetic acid than is the brain enzyme (K_i = 1·10^?6 M); (3) brain but not renal glutamate decarboxylase activity can be substantially stimulated in vitro by the addition of exogenous pyridoxal 5′-phosphate; (4) renal glutamate decarboxylase is significantly decreased in renal cortex from rats on a low-salt diet. Proximal tubules are enriched in glutamate decarboxylase compared to the activity in whole renal cortex or glomeruli (42, 22 and 14 nmols/mg protein per h, respectively). We speculate that renal γ-aminobutyric acid synthesis does not reflect the presence of GABAergic renal nerves, but may serve a function in proximal tubular cells.     

Interleukin-19 Mediates Tissue Damage in Murine Ischemic Acute Kidney Injury

Inflammation and renal tubular injury are major features of acute kidney injury (AKI). Many cytokines and chemokines are released from injured tubular cells and acts as proinflammatory mediators. However, the role of IL-19 in the pathogenesis of AKI is not defined yet. In bilateral renal ischemia/reperfusion injury (IRI)-induced and HgCl₂-induced AKI animal models, real-time quantitative (RTQ)-PCR showed that the kidneys, livers, and lungs of AKI mice expressed significantly higher IL-19 and its receptors than did sham control mice. Immunohistochemical staining showed that IL-19 and its receptors were strongly stained in the kidney, liver, and lung tissue of AKI mice. In vitro, IL-19 upregulated MCP-1, TGF-β1, and IL-19, and induced mitochondria-dependent apoptosis in murine renal tubular epithelial M-1 cells. IL-19 upregulated TNF-α and IL-10 in cultured HepG2 cells, and it increased IL-1β and TNF-α expression in cultured A549 cells. In vivo, after renal IRI or a nephrotoxic dose of HgCl₂ treatment, IL-20R1-deficient mice (the deficiency blocks IL-19 signaling) showed lower levels of blood urea nitrogen (BUN) in serum and less tubular damage than did wild-type mice. Therefore, we conclude that IL-19 mediates kidney, liver, and lung tissue damage in murine AKI and that blocking IL-19 signaling may provide a potent therapeutic strategy for treating AKI.     

Dual-enzyme assay of glutamate in single cells based on capillary electrophoresis

A new dual-enzyme on-column reaction method combined with capillary electrophoresis has been developed for determining the glutamate content in single cells. Glutamate dehydrogenase and glutamic pyruvic transaminase were used to catalyze the glutamate reaction. Detection was based on monitoring the laser-induced fluorescence of the reaction product NADH, and the measured fluorescence intensity was related to the concentration of glutamate in each cell. Glutamate dehydrogenase catalyzed the formation of NADH, and glutamic pyruvic transaminase drives the glutamate dehydrogenase reaction by removing a reaction product and regenerating glutamate. The detection limit of glutamate is down to the 10⁻⁸ M level, which is 1 order of magnitude lower than previously reported detection limits based on similar detection methods. The mass detection limit of a few attomoles is far superior to that of any other reports. Selectivity for glutamate is excellent over most amino acids. The glutamate content in single human erythrocytes and baby rat brain neurons were determined with this method and the results agreed well with literature values.