The chlorinated lipidome originating from myeloperoxidase-derived HOCl targeting plasmalogens: Metabolism, clearance, and biological properties.

Myeloperoxidase produces the two-electron oxidant HOCl, which targets plasmalogen phospholipids liberating 2-chlorofatty aldehyde. 2-Chlorofatty aldehyde has four known fates: 1) oxidation to 2-chlorofatty acid; 2) reduction to 2-chlorofatty alcohol; 3) Schiff base adduct formation with proteins and amines; and 4) reactivity with glutathione through nucleophilic attack of the α-chlorinated carbon. 2-Chlorofatty acid does not undergo conventional fatty acid ß-oxidation due to the presence of the α-chlorinated carbon; however, 2-chlorofatty acid does undergo sequential ω-oxidation and ß-oxidation from the ω-end, ultimately resulting in 2-chloroadipic acid urinary excretion. Recent studies have demonstrated that 2-chlorofatty acid clearance is increased by treatment with the PPAR-α agonist WY14643, which increases the enzymatic machinery responsible for hepatic ω-oxidation. Furthermore, 2-chlorofatty acid has been shown to be a PPAR-α agonist, and thus accelerates its own clearance. The roles of 2-chlorofatty aldehyde and 2-chlorofatty acid on leukocyte and endothelial function have been explored by several groups, suggesting that chlorinated lipids induce endothelial cell dysfunction, neutrophil chemotaxis, monocyte apoptosis, and alterations in vascular tone. Thus, the chlorinated lipidome, produced in response to leukocyte activation, is a potential biomarker and therapeutic target to modulate host response in inflammatory diseases.

Protective effects of L-type fatty acid-binding protein (L-FABP) in proximal tubular cells against glomerular injury in anti-GBM antibody-mediated glomerulonephritis.

BACKGROUND: In glomerulonephritis (GN), an overload of free fatty acids (FFA) bound to albumin in urinary protein may induce oxidative stress in the proximal tubules. Human liver-type fatty acid-binding protein (hL-FABP) expressed in human proximal tubules, but not rodents, participates in intracellular FFA metabolism and exerts anti-oxidative effects on the progression of tubulointerstitial damage. We examined whether tubular enhancement of this anti-oxidative action modulates the progression of glomerular damage in immune-mediated GN in hL-FABP chromosomal gene transgenic (Tg) mice. METHODS: Anti-glomerular basement membrane antibody-induced glomerulonephritis (anti-GBM GN) was induced in Tg and wild-type mice (WT). Proteinuria, histopathology, polymorphonuclear (PMN) influx, expression of tubulointerstitial markers for oxidative stress 4-hydroxy-2-Nonenal (HNE) and fibrosis (α-smooth muscle actin), proximal tubular damage (Kim-1), Peroxisome Proliferator-Activated Receptor γ (PPAR γ) and inflammatory cytokines [Monocyte Chemotactic Protein-1, tumor necrosis factor-alpha (TNF-α) and Transforming growth factor beta (TGF-ß)] were analyzed. The mice were also treated with an angiotensin type II receptor blocker (ARB). RESULTS: The urinary protein level in Tg mice decreased significantly during the acute phase (~Day 5). Tg mice survived for a significantly longer time than WT mice, with an attenuation of tubulointerstitial damage score and expression of each tubulointerstitial damage marker observed at Day 7. Expression of inflammatory cytokines on Day 7 was higher in WT mice than Tg mice and correlated strongly with PPARγ expression in WT mice, but not in Tg mice. Interestingly, Tg mice showed insufficient PMN influx at 3 and 6 h, with simultaneous elevation of urinary L-FABP and reduction in HNE expression. The two strains of mice showed different types of glomerular damage, with mild mesangial proliferation in Tg mice and severe endothelial swelling with vascular thrombosis in WT mice. The glomerular damage in Tg mice was improved by administration of an ARB. CONCLUSIONS: The present experimental model suggests that tubular enhancement of L-FABP may protect mice with anti-GBM GN from progression of both tubulointerstitial and glomerular injury.

Urinary fatty acids and liver-type fatty acid binding protein in diabetic nephropathy.

BACKGROUND: The aims of this clinical study were to investigate the associations of urinary free fatty acid (FFA) levels with tubulointerstitial damage, and to determine the clinical significance of urinary liver-type fatty acid binding protein (L-FABP) in diabetic nephropathy. METHODS: Fifteen patients with nephrotic syndrome due to diabetic nephropathy and 12 patients with minimal-change nephrotic syndrome (MCNS) were studied. Urinary and serum FFA concentrations (palmitic, oleic, linoleic, and arachidonic acids) were measured by gas chromatography, and urinary L-FABP levels were quantified using an ELISA technique. Tubulointerstitial damage was assessed using renal biopsy specimens. RESULTS: The levels of urinary linoleic and arachidonic acids were significantly elevated in diabetic nephropathy compared to MCNS patients, though serum FFA levels were lower in diabetic nephropathy than MCNS patients. The degree of tubulointerstitial damage was significantly severer in the patients with diabetic nephropathy than MCNS. Urinary L-FABP and 8-OHdG (8-hydroxydeoxyguanosine) concentrations were significantly higher in the diabetic nephropathy subjects. CONCLUSION: Elevated urinary excretion of FFA may be a reflection of FFA overload in the proximal tubules, and FFA may be an important promoter of tubulointerstitial damage in diabetic nephropathy patients. Urinary L-FABP levels may reflect the stress induced by FFA to the proximal tubules, leading to severe tubulointerstitial damage.

Metabolomic study of cisplatin-induced nephrotoxicity.

We have shown that cisplatin inhibits fatty acid oxidation, and that fibrate treatment ameliorates renal function by preventing the inhibition of fatty acid oxidation and proximal tubule cell death. Urine samples of mice treated with single injection of cisplatin (20 mg/kg body weight) were collected for 3 days and analyzed by 1H-nuclear magnetic resonance (NMR) spectroscopy. In a separate group, urine samples of mice treated with peroxisome proliferator-activated receptor-alpha (PPARalpha) ligand WY were also analyzed by NMR after 2 days of cisplatin exposure. Biochemical analysis of endogenous metabolites was performed in serum, urine, and kidney tissue. Electron microscopic studies were carried out to examine the effects of PPARalpha ligand and cisplatin. Principal component analysis demonstrated the presence of glucose, amino acids, and trichloacetic acid cycle metabolites in the urine after 48 h of cisplatin administration. These metabolic alterations precede changes in serum creatinine. Biochemical studies confirmed the presence of glucosuria, but also demonstrated the accumulation of nonesterified fatty acids, and triglycerides in serum, urine, and kidney tissue, in spite of increased levels of plasma insulin. These metabolic alterations were ameliorated by the use of PPARalpha ligand. Electron microscopic analysis confirmed the protective effect of the fibrate on preventing cisplatin-mediated necrosis of the S3 segment of the proximal tubule. Our study shows that cisplatin-induces a unique NMR metabolic profile in urine of mice that developed acute renal failure, and confirms the protective effect of a fibrate class of PPARalpha ligands. We propose that the injury-induced metabolic profile may be used as a biomarker of cisplatin-induced nephrotoxicity.

Urinary free fatty acids bound to albumin aggravate tubulointerstitial damage.

BACKGROUND: Evidence indicates that urinary protein is associated with tubulointerstitial damage and thus it is an aggravating factor for chronic renal disease. As free fatty acids (FFAs) are bound to serum albumin, we hypothesized that FFAs were overloaded to the proximal tubule in massive proteinuria and thus caused tubulointerstitial damage. To test this hypothesis, massive proteinuria was provoked in mice and the renal damage examined. METHODS: Mice were intraperitoneally injected with bovine serum albumin (BSA) replete with FFAs (r-BSA group, N = 10), FFA-depleted BSA (d-BSA group, N = 10), or saline (saline group, N = 9) for 14 days. RESULTS: The kidneys of the r-BSA group showed severe tubulointerstitial damage and those of the d-BSA group showed mild tubulointerstitial damage. Urinary excretion of both total protein and mouse albumin were significantly higher in the r-BSA group than in the d-BSA group. To examine the proximal tubular uptake of albumin, the BSA content in the cultured mouse proximal tubules was measured by ELISA after 90 minutes of incubation with each BSA. In terms of the BSA content in the proximal tubules, there was no significant difference between the r-BSA and the d-BSA groups. These results indicate that r-BSA and d-BSA were similarly reabsorbed into the proximal tubule and that r-BSA causes severe tubulointerstitial damage. CONCLUSIONS: It is the FFAs bound to albumin, rather than albumin itself, which cause severe tubulointerstitial damage by being reabsorbed into the proximal tubule. To our knowledge, this is the first in vivo observation in which FFAs have caused severe tubulointerstitial injury.

Urinary excretion of biopyrrins, oxidative metabolites of bilirubin, increases after spasm provocation tests in patients with coronary spastic angina.

BACKGROUND: Bilirubin apparently functions as an antioxidant in vivo by reacting with reactive oxygen species, and, as a result, becomes oxidized. The urinary excretion of oxidative metabolites of bilirubin, biopyrrins, could be a biological marker for in vivo production of reactive oxygen species. The purpose of this study was to examine the extent of oxidative stress in patients with possible ischemic heart diseases (n=44) by measuring urinary biopyrrins by enzyme-linked immunosorbent assay before and after the spasm provocation test (SPT). METHODS: Spot urine samples were collected five times; 1 day before, in the morning just before, immediately after, 6 h after, and 1 day after the SPT. Nineteen patients were positive to SPT judged from the specific changes in electrocardiogram for myocardial ischemia following intracoronary injections of ergonovine. RESULTS: The baseline data such as age, sex, number of risk factors and concentrations of serum bilirubin, and the measured hemodynamic parameters of heart rate, blood pressure, left ventricular end-diastolic pressure and left ventricular ejection fraction were not different between the positive and negative groups. The baseline concentrations of biopyrrins during the control period were not significantly different between the two groups. However, they increased significantly after the SPT, thereby the magnitude of increases immediately after and 6 h after the SPT were significantly (P<0.001 and P<0.01, respectively) greater in the positive group than in the negative. CONCLUSION: The present findings strongly suggest that coronary arterial occlusion augments production of biopyrrins, which indicates exposure to oxidative stress in patients with ischemic heart diseases.

Pitfalls in the use of 2-octynoic acid as an in vivo model of medium-chain acyl-coenzyme A dehydrogenase deficiency: ketone turnover and metabolite studies in the rat.

2-Octynoic acid was administered by intraperitoneal injection to fasted Sprague-Dawley rats in an attempt to simulate medium-chain acyl-coenzyme A dehydrogenase (MCAD) deficiency. The resultant urine organic acid profile showed a mild dicarboxylic aciduria but lacked the glycine conjugates characteristic of MCAD deficiency. Further studies with infused 13C(4)-3-hydroxybutyrate and 13C(2)-acetoacetate demonstrated reduced ketone production in treated rats compared with control animals. Although plasma ketone body concentrations were low in treated rats, plasma free fatty acids were also low, thereby providing diminished substrate for ketone production. This is the reverse of the finding in children with MCAD deficiency, who have low levels of plasma ketones despite elevated free fatty acids. These animal studies were therefore not helpful in improving our understanding of ketone body kinetics in children with MCAD deficiency.

Identification of 9,10-epoxyoctadecanoic acid in human urine using gas chromatography-mass spectrometry.

9,10-epoxyoctadecanoic acid has been detected in human urine. Two simple purification procedures were used; the one based upon liquid-liquid extraction and the other based upon sorbent extraction technology isolating the free fatty acid fraction. Prior to trimethylsilylation and gas chromatographic-mass spectrometric analysis, both the epoxy and carboxy functions were reduced to hydroxy groups. The shift in fragmentation of a deuterated sample verified the presence of intact epoxide prior to chemical reduction. Special attention was paid to the risk of false identification of the epoxide. The content of 9,10-epoxyoctadecanoic acid in human urine was estimated to be 2.1 nM/L.

Sequential determination of triglycerides and free fatty acids in biological fluids by use of a continuous pretreatment module coupled to a gas chromatograph.

A continuous system coupled to a gas-liquid chromatograph was used for the sequential determination of triglycerides and free fatty acids in serum and urine. The module provides compositional information and hence more detailed information on lipid metabolism changes in patients suffering metabolic disorders. Lipids in biological samples are manually extracted in methanol-n-hexane and introduced into the flow system; free fatty acids are then separated by retention on an ion-exchange resin and triglycerides (not retained) are transesterified with acetyl chloride in methanol. The resulting methyl esters are continuously injected into the gas chromatograph and determined by using a flame ionization detector. In a second step, retained free fatty acids are eluted and derivatized (also with acetyl chloride in methanol) and subsequently determined similarly as the triglycerides. The proposed method was applied to the determination of triglycerides in a lipid control serum; free fatty acids were determined in a human pool serum by the proposed method and compared with the volumetric method used in clinical practice. The results obtained in both instances showed good agreement between the results provided for triglycerides and free fatty acids. The proposed method was also applied to urine samples; a parallel recovery study was also made in order to assess the performance of the method.

Biochemical effects of Catha edulis, cathine and cathinone on adrenocortical functions.

Catha edulis is a plant that grows in certain areas of East Africa and the Arab Peninsula. The stimulating properties of fresh material were described more than seven centuries ago and today the habit of inducing a state of euphoria and subjective well-being by chewing Catha edulis prevails among the inhabitants of these regions. Oral administration of this plant and its active constituents (cathine and cathione) on experimental animals might have stimulating effects on adrenocortical function. This was indicated by the significant decrease in adrenal cholesterol, ascorbic acid, glycogen, and the increase in adrenal phosphorylase activity. In addition, the level of urinary 17-hydroxycorticosteroids and plasma free fatty acids were increased.

2,4-Dienoyl-coenzyme A reductase deficiency: a possible new disorder of fatty acid oxidation.

Several inherited disorders of fatty acid beta-oxidation have been described that relate mainly to saturated precursors. This study is the first report of an enzyme defect related only to unsaturated fatty acid oxidation and provides the first in vivo evidence that fat oxidation in humans proceeds by the reductase-dependent pathway. The patient was a black female, presenting in the neonatal period with persistent hypotonia. Biochemical studies revealed hyperlysinemia, hypocarnitinemia, normal organic acid profile, and an unusual acylcarnitine species in both urine and blood. The new metabolite was positively identified by mass spectrometry as 2-trans,4-cis-decadienoylcarnitine, derived from incomplete oxidation of linoleic acid. In spite of dietary therapy, the patient died of respiratory acidosis at four months of age. Samples of liver and muscle from the autopsy were assayed for 2,4-dienoyl-coenzyme A reductase activity. Using the substrate 2-trans,4-cis-decadienoylcoenzyme A, the reductase activity was 40% of the control value in liver and only 17% of that found in normal muscle. It is suggested that unsaturated substrates should be used for in vitro testing to cover the full range of potential beta-oxidation defects and that acylcarnitine species identification be used for in vivo detection of this disorder.

The metabolic effects of acute and chronic administration of gastrozepin.

The structure of gastrozepin (G) resembles that of tricyclic antidepressants and antihistaminic and antiserotonic cyproheptadine which are compounds with known metabolic effects. The object of this study was to test the potential action of G on the metabolism of basic nutrients, insulin and selected hormonal parameters after parenteral administration (10 and 20 mg G) and after chronic oral administration (50 mg G) during antiulcer therapy. Investigations carried out in two groups of 7 and 9 healthy volunteers, respectively, and on one group of seven patients with ulcer disease, revealed no changes in any of the blood parameters studied, nor in the excretion of catecholamines or 17-OH steroids in the urine. Our observations practically rule out any metabotropic activity of G, thereby differentiating it from tricyclic antidepressants and cyproheptadine. Isolated significant differences from the basal value seen after G and saline, can be attributed to other than pharmacological or specific mechanisms such as stress associated with the first examination, effect of fasting, individual response of the subjects, and so on.

Blood and urinary magnesium, zinc, calcium, free fatty acids, and catecholamines in type A and type B subjects.

Twenty type A male students were compared to nineteen type B male students (all in apparently good health), before and after exposure to combined stress (noise and task). Before stress, red blood cell (RBC) Zn concentration is higher (P less than .05) and Zn excretion lower (P less than .05) in type A than in type B people. After stress, type A subjects exhibit changes that are larger and more significant than those of type B individuals. After stress, the type A group shows an important increase of urinary catecholamines (P = 2.10(-5), serum free fatty acids, and urinary Zn (P = .001); a slight increase in plasma magnesium (P less than .05); and a small but significant decrease in RBC Mg (P less than .02). These results suggest that type A subjects are more sensitive to stress than are type B people and more readily lose their intracellular Mg, the rise in plasma Mg being a transient one, probably consecutive to the cellular loss. The present observations are in good agreement with published data: ie, the psychological characteristics of type A personalities; their greater susceptibility to ischemic heart disease, which has been associated with Mg deficiency; the possible role of hypomagnesemia in the pathogenesis of hypertension and coronary vasospasm; and the high RBC Zn levels found in hypertensive patients.

Vagotonicity of violence: biochemical and cardiac responses to violent films and television programmes.

In a search for a reproducible means of evoking different types of emotional stress it was found that in spite of increased adrenaline secretion slowing of the heart occurred when watching violent television programmes. Further evidence of increased vagal tone was provided by the "sinus arrhythmia" effect, a widening of the gap between the maximum and minimum heart rates during the respiratory cycle in parts of the humour, violence, and suspense sections of the television programme.Groups of people taken to see two particularly violent films showed similar evidence suggesting vagal overactivity, together with increases in plasma free fatty acids and decreases in triglycerides. As these changes occurred even with beta-blockade it is suggested that they might be caused by non-sympathetically mediated changes in the levels of hormones, such as growth hormone, producing lipolysis.The ability to assess objectively an individual's reaction to viewing violence might make it possible to judge the likely social impact of violent films and television programmes.