Synovial fluid lactic acid in septic arthritis

Lactic acid concentrations in the synovial fluid of 71 patients with inflammatory arthritis were determined by an enzyme method. In 63 samples from 54 patients with a variety of non-septic arthritides, including rheumatoid arthritis, reactive arthritis and gout, the concentration of lactic acid was never greater than 10.2 mmol/l, whereas all twelve patients with septic arthritis had concentrations of 11 mmol/l or greater. Two patients with gonococcal arthritis did not have raised lactic acid concentrations. The enzyme method of lactic acid estimation is an accurate reproducible means of differentiating septic from nonseptic arthritis prior to the isolation of the infecting organism. However, caution is necessary when interpreting the results in those patients who have recently received antibiotic therapy, or in whom gonococcal arthritis is suspected.     

Quantitation of the fatty acid composition of phosphatidic acid by capillary gas chromatography electron-capture detection with picomole sensitivity

We describe a relatively simple and sensitive method to measure femtomole amounts of phosphatidic acid in cells. Phosphatidic acid was extracted from cells in the presence of 1-heptadecanoyl-2-heptadecanoyl-sn-glycero-3-phosphate as an internal standard, purified by two-dimensional thin-layer chromatography, and hydrolyzed to its constituent free fatty acids which were then derivatized to the corresponding pentafluorobenzyl esters. Pentafluorobenzyl esters of fatty acids were analyzed by gas chromatography with electron-capture detection. Long-chain fatty acids were resolved with excellent signal-to-noise ratios. Using heptadecanoic acid as an internal standard for quantitation, as little as 1 fmol of pentafluorobenzyl ester of stearic acid was detected with a linear response up to 10 pmol. Linear detector responses were obtained for all major classes of fatty acids. For phosphatidic acid measurement, the detection limit was at least 50 fmol thus achieving a 1000-fold increase in sensitivity compared to the most sensitive of the previously described methods. An example is provided of quantitating phosphatidic acid from minute amounts of biological samples such as islets of Langerhans.     

A new HPLC-based method for the quantitative analysis of inner stratum corneum lipids with special reference to the free fatty acid fraction

The inner stratum corneum is likely to represent the location of the intact skin barrier, unperturbed by degradation processes. In our studies of the physical skin barrier a new high-performance liquid chromatography (HPLC)-based method was developed for the quantitative analysis of lipids of the inner stratum corneum. All main lipid classes were separated and quantitated by HPLC/light scattering detection (LSD) and the free fatty acid fraction was further analysed by gas-liquid chromatography (GLC). Mass spectrometry (MS) was used for peak identification and flame ionization detection (FID) for quantitation. Special attention was paid to the free fatty acid fraction since unsaturated free fatty acids may exert a key function in the regulation of the skin barrier properties by shifting the physical equilibrium of the multilamellar lipid bilayer system towards a noncrystalline state. Our results indicated that the endogenous free fatty acid fraction of the stratum corneum barrier lipids in essence exclusively consisted of saturated long-chain free fatty acids. This fraction was characterized as a very stable population (low interindividual peak variation) dominated by saturated lignoceric acid (C24:0, 39 molar%) and hexacosanoic acid (C26:0, 23 molar%). In addition, trace amounts of very long-chain (C32-C36) saturated and monounsaturated free fatty acids were detected in human forearm inner stratum corneum. Our analysis method gives highly accurate and precise quantitative information on the relative composition of all major lipid species present in the skin barrier. Such data will eventually permit skin barrier model systems to be created which will allow a more detailed analysis of the physical nature of the human skin barrier.     

Comparison of fatty acid composition of stable L-phase variants of Staphylococcus aureus induced by three differnet mechanisms

The total saponifiable fatty acids of three stable L-phase variants of Staphylococcus aureus induced by cycloserine, methicillin, and lysostaphin were examined by gas-liquid chromatography. Five separate preparations of each of the three variants were examined. Twenty-nine fatty acids were identified. The fatty acid patterns of the three variants were very similary.     

Fatty acid composition of myelin proteolipid protein during vertebrate evolution

The hydrophobic myelin proteolipid protein (PLP) contains covalently bound long-chain fatty acids which are attached to intracellular cysteine residues via thioester linkages. To gain insight into the role of acylation in the structure and function of myelin PLP, the amount and pattern of acyl groups attached to the protein during vertebrate evolution was determined. PLP isolated from brain myelin of amphibians, reptiles, birds and several mammals was subjected to alkaline methanolysis and the released methyl esters were analyzed by gas-liquid chromatography. In all species studied, PLP contained approximately the same amount of covalently bound fatty acids (3% w/w), and palmitic, palmitoleic, oleic and stearic acids were always the major acyl groups. Although the relative proportions of these fatty acids changed during evolution, the changes did not necessarily follow the variations in the acyl chain composition of the myelin free fatty acid pool, suggesting fatty acid specificity. The phylogenetic conservation of acylation suggests that this post-translational modification is critical for PLP function.     

Changes in plasma and colonic mucosa fatty acid profiles in rats with ulcerative colitis induced by trinitrobenzene sulfonic acid

Polyunsaturated fatty acids have a key role in the pathogenesis of inflammatory bowel disease since some of the arachidonic acid-derived eicosanoids have been found to be increased in inflamed intestinal mucosa in the acute phase of human disease. The aim of this study was to prospectively assess plasma and colon mucosa fatty acid patterns in rats with experimental ulcerative colitis. Twenty rats were treated with trinitrobenzene sulfonic acid and 20 with NaCl; two groups were killed after one week and two after two weeks to evaluate colon damage. Plasma was obtained by aortic puncture and colonic mucosa was scraped off and the fatty acid pattern was determined by gas-liquid chromatography. Total, saturated, and monounsaturated plasma fatty acids were significantly higher in both periods of ulcerative colitis as compared to controls. Plasma n-6 fatty acids were increased after treatment, but no significant changes were observed concerning to n-3 fatty acids. With regard to colon mucosa, saturated and monounsaturated fatty acids did not change because of the disease; however, n-6 fatty acids decreased in the first week and increased in the second week and n-3 fatty acids were increased. Changes on the fatty acid distribution in plasma did not parallel to those of colonic mucosa except for 22:6(n-3). We have also found that experimental ulcerative colitis induced by trinitrobenzene sulfonic acid reproduces many of the features related to changes in plasma and colon mucosa fatty acids observed in the human disease.     

Fatty acid desaturase activities and polyunsaturated fatty acid composition in human liver between the seventeenth and thirty-sixth gestational weeks

OBJECTIVE: The aim of the study was to characterize n-3 and n-6 fatty acid delta5- and delta6-desaturase activities and their time course variations in human fetal liver between the 17th and 36th gestational week. STUDY DESIGN: Twenty-one biologic samples were obtained after legally approved medical abortion, according to French law. The desaturase activities were measured in the 21 liver samples by a radiochemical method by means of reverse-phase high-performance liquid chromatography. The fatty acid composition (percentage by weight) of liver phospholipids was assessed in 16 samples by gas-liquid chromatographic analysis. RESULTS: Both delta5- and delta6-desaturase activities were significantly expressed between the 17th and 36th gestational weeks. During the second trimester n-6 fatty acid delta5- and delta6-desaturase activities showed opposite patterns of variation; both then remained stable between the 25th and 36th weeks. Delta6-desaturation was higher in n-3 than n-6 fatty acids and peaked at the 18th gestational week. The percentages of linoleic and docosahexaenoic acids in liver microsomes were positively correlated with the gestation age (P < .01), whereas arachidonic acid remained stable. CONCLUSION: Significant n-3 and n-6 delta5- and delta6-desaturase activities are expressed in human fetal liver as early as the 17th gestational week and are stable throughout the third trimester. Their theoretic capacity evaluated from in vitro measurements appears lower than polyunsaturated fatty acid requirements and is not directly related to liver microsomal membrane fatty acid composition.     

High-pressure liquid chromatographic analysis of drugs in biological fluids. IV. Determination of clofibrinic acid

A rapid, sensitive and specific high-pressure liquid chromatographic method is described for the quantitative analysis of clofibrinic acid in plasma, saliva and urine. In contrast to previously reported gas-liquid chromatographic methods, which require derivatization of clofibrinic acid before chromatography, the present method involves a simple two-step extraction procedure and chromatographic determination of the underivatized clofibrinic acid. Concentrations between 1.0 and 25.0 microgram per sample can be measured with a coefficient of variation from 1 to 6%.     

Free fatty acids in gallbladder bile

Using gas chromatography and high performance liquid chromatography (HILC), we examined free fatty acid and lecithin molecular species in gallbladder biles from patients with cholesterol gallstones. Effect of free fatty acids on cholesterol nucleation in model bile was studied by a sensitive cholesterol crystal growth assay. Compared to bile of controls, biles from patients with gallstones had higher total free fatty acid level, more palmitic acid, more stearic acid, more linoleic acid and arachidonic acid. The lecithin pattern was similar in all. After free fatty acids were added to model bile, palmitic acid, oleic, acid, linoleic acid and arachidonic acid had significant effect of pro-nucleating, free fatty acids on non-protein pro-nucleating factor. These data suggest that variations in quantitation and composition of free fatty acids are importanct in the pathogenesis of cholesterol gallstone formation.     

Evaluation of sodium hyaluronate therapy in induced septic arthritis in the horse

This study was conducted to determine the efficacy of sodium hyaluronate (SH) with antibiotic therapy and joint lavage for reducing acute inflammatory and degenerative changes induced by septic arthritis. Septic arthritis was induced in six adult horses by inoculating the tarsocrural joints with 1 x 10(4) colony-forming units of Staphylococcus aureus. When clinical signs appeared, trimethoprim-sulphamethoxazole (30 mg/kg bodyweight [bwt] daily) and phenylbutazone (4.4 mg/kg bwt sid) were administered and continued until termination of the study (Treatment Day 18). Twenty-four hours post inoculation, all joints were lavaged with sterile lactated Ringer's solution. Following lavage, one joint of each horse was injected with 10 mg of SH, and the contralateral joint served as the control. Sodium hyaluronate treated joints showed significant reductions in lameness, tarsal circumference and synovial fluid protein and WBC concentrations. The synovial membrane of the SH-treated joints contained less cellular infiltrate, less granulation tissue formation and retained a more normal villous structure compared with controls. The total glycosaminoglycan loss from the articular cartilage in the SH treated joints was consistently less than that from the control joints; however, this difference was not statistically significant. Sodium hyaluronate with joint lavage appears to be more beneficial than lavage alone for treatment of septic arthritis.     

Septic arthritis caused by Streptococcus pneumoniae

BACKGROUND: Streptococcus pneumoniae is an uncommon agent of infective arthritis. In this report three cases of pneumococcal arthritis are described. METHODS: Retrospective review of synovial fluids processed in our laboratory yielding bacteria. The study period was from January 1991 to December 1995. The clinical records of patients with the clinical and microbiological diagnosis of septic arthritis were reviewed. RESULTS: Twenty-eight out of a total of 43 clinical records had the clinical and microbiological diagnosis of septic arthritis and three (11%) were caused by Streptococcus pneumoniae. The infective source in two of these three cases was probably the respiratory tract, and the most common location was the knee. CONCLUSIONS: In our cases immunosuppression seemed to be the major risk factor involved in the development of pneumococcal arthritis.     

Elevated nerve growth factor levels in the synovial fluid of patients with inflammatory joint disease

A novel pH shock extraction procedure was used to measure nerve growth factor (NGF) levels in both normal and inflamed synovial fluids using a sensitive and specific two-site enzyme linked immunosorbant assay. To date no data is available on NGF levels in normal synovial fluids. Synovial fluids were taken from 5 normal volunteers, 12 patients with rheumatoid arthritis and 10 patients with other inflammatory arthropathies. The mean +/- SEM NGF concentration in normal synovial fluids was 95 +/- 33.2 pg/ml (range 39.1-143.1 pg/ml), whereas the mean NGF concentration in the synovial fluids taken from patients with rheumatoid arthritis was 532.5 +/- 123.8 pg/ml (range 152-1686 pg/ml). The mean NGF concentration in patients with other inflammatory arthropathies was also raised (430.6 +/- 90 pg/ml; range 89-1071 pg/ml). The NGF concentrations were significantly higher in the synovial fluids from both inflamed groups (ANOVA p < 0.05) compared to normals. Raised levels of NGF in synovial fluid may contribute directly to joint inflammation via activation of inflammatory cells.     

Trans fatty acid content of human milk in Spain

The C18 fatty acid fraction of 38 samples of mature human milk from Spanish women was analyzed using capillary gas chromatography. The average content of trans fatty acids found in these samples represented 0.95% of total fatty acids. This value is lower than the percentage found in human milk from other developed countries in which the consumption of hydrogenated fat is higher. Trans fatty acid content in human milk has been related to the types of fats and oils present in the diets of the nursing mothers. According to the results obtained in this survey, we can also conclude that the amount of trans forms in breast milk lipids is lower than the trans content found in infant formulas in Spain.     

Fatty acid composition of myelin isolated from the brain of a patient with cellular deficiency of co-enzyme forms of vitamin B12

Myelin fractionation and subsequent lipid isolation have been carried out on a brain from a patient who suffered from a cellular deficiency of the adenosylcobalamin and methylcobalamin co-enzyme forms of vitamin B12. Examination of the fatty acid composition of choline and ethanolamine glycerophospholipids indicated a relative enrichment of odd-chain fatty acids which were identified by gas-liquid chromatography-mass spectroscopy as C15, C15:1, C17 and C17:1. A mixture of methyl branched C17 fatty acids was also identified. Odd-chain fatty acids accounted for 9.8% of the total fatty acid in the myelin choline phospholipid conpared to control values of 1.2%. The affected brain myelin phospholipids had a lower unsaturated fatty acid content. Examination of the myelin sphingolipids, sphingomyelin, cerebroside and sulfatide, yielded abnormal fatty acid profiles. The sphingomyelin contained only small amounts of C24:1 fatty acid. Both normal and hydroxy fatty acid containing cerebroside and sulfatide had reduced levels of C24 fatty acid. Determination of the relative hydroxy and normal fatty acid content of the galactolipids indicated an abnormally high hydroxy fatty acid level. Abnormal fatty acid profiles of brain cerebral sphingolipids have not been previously described in cases of vitamin B12 deficiency. Whether or not these alterations are characteristic will only be established by estimating sphingolipids in other such cases.     

Tumor necrosis factor-induced release of endogenous fatty acids analyzed by a highly sensitive high-performance liquid chromatography method

A highly sensitive method to determine agonist-induced release of endogenous fatty acids from cells in culture was developed using high-performance liquid chromatography and fluorescence detection. Fatty acids were selectively derivatized with 1-pyrenyldiazomethane and separated on a LC18 reversed phase column using an acetonitrile-water gradient. The detection limit was approx. 20 fmol and the recovery of the complete method using oleic acid was 93-98%. Tumor necrosis factor alpha (TNF-alpha) increased the extracellular release of endogenous arachidonic acid (20:4n-6) from 21 to 153 pmol/well per 4 h using 2.7 x 10(6) WEHI fibrosarcoma cells. In cells preincubated with 50 microM 20:4n-6, the corresponding figures were 463 and 3379 pmol 20:4n-6/well. Simultaneously, nearly equimolar amounts of 22:4n-6 were released together with slightly lower amounts of 24:4n-6, 16:0, 16:1n-9, and 18:1n-9. Analysis of cell lipid fatty acids showed that phosphatidylcholine was the major source of the released fatty acids. TNF-alpha increased the intracellular concentration of unesterified 20:4n-6 and 22:4n-6 by 368% and 451%, respectively. This suggests that released 20:4n-6 is rapidly chain elongated to 22:4n-6. The results indicate that the present method facilitates studies on agonist-induced release of endogenous fatty acids, and that TNF-induced fatty acid release seems to be less selective for 20:4n-6 than previously reported.     

Liver microsomal membrane fluidity and microsomal desaturase activities in adult spontaneously hypertensive rats

OBJECTIVE: The purpose of the present study was to investigate liver microsomal membrane fluidity simultaneously with membrane fatty acid composition and desaturase activities in spontaneously hypertensive rats (SHR). DESIGN AND METHODS: The membrane fluidity was determined, after electron spin resonance (ESR) measurement, in SHR compared with normotensive Wistar-Kyoto (WKY) rats, by calculating the order parameter S from ESR spectra of 5-nitroxide stearate and 10-nitroxide stearate, used as spin-labelled fatty acids. Desaturase activities were measured by incubating SHR and WKY rat liver microsomes with [14C]-radiolabeled fatty acids as substrates for desaturation reactions. The fatty acid composition of liver microsomal membranes was determined by gas-liquid chromatography. RESULTS: Whereas no significant difference between S of 5-nitroxide stearate was observed for SHR and WKY rats, S of 10-nitroxide stearate was significantly lower in SHR than it was in WKY rat microsomal membrane, indicating that the core microsomal membrane fluidity was higher in SHR. Significant differences between fatty acid compositions were observed for SHR and WKY rat microsomal membranes. Delta9 and n-6 delta6 microsomal desaturase activities were significantly lower in SHR. CONCLUSION: These results suggest that the higher liver core microsomal membrane fluidity observed in SHR might be dependent on the increased proportion of mono-unsaturated fatty acids. Such observed modifications and the alterations in delta9 and n-6 delta6 desaturase activities suggest that an impaired polyunsaturated fatty acid biosynthesis is related to changes in microsomal membrane fluidity in hypertension.     

Intra-arterial fluorescein angiography using ophthalmic artery catheter in an experimental model

BACKGROUND: Borrelia burgdorferi is difficult to detect in synovial fluid, which limits our understanding of the pathogenesis of Lyme arthritis, particularly when arthritis persists despite antibiotic therapy. METHODS: Using the polymerase chain reaction (PCR), we attempted to detect B. burgdorferi DNA in joint-fluid samples obtained over a 17-year period. The samples were tested in two separate laboratories with four sets of primers and probes, three of which target plasmid DNA that encodes outer-surface protein A (OspA). RESULTS: B. burgdorferi DNA was detected in 75 of 88 patients with Lyme arthritis (85 percent) and in none of 64 control patients. Each of the three OspA primer-probe sets was sensitive, and the results were moderately concordant in the two laboratories (kappa = 0.54 to 0.73). Of 73 patients with Lyme arthritis that was untreated or treated with only short courses of oral antibiotics, 70 (96 percent) had positive PCR results. In contrast, of 19 patients who received either parenteral antibiotics or long courses of oral antibiotics (> or = 1 month), only 7 (37 percent) had positive tests (P < 0.001). None of these seven patients had received more than two months of oral antibiotic treatment or more than three weeks of intravenous antibiotic treatment. Of 10 patients with chronic arthritis (continuous joint inflammation for one year or more) despite multiple courses of antibiotics, 7 had consistently negative tests in samples obtained three months to two years after treatment. CONCLUSIONS: PCR testing can detect B. burgdorferi DNA in synovial fluid. This test may be able to show whether Lyme arthritis that persists after antibiotic treatment is due to persistence of the spirochete.     

Clear cell sarcoma of soft tissues. Clinico-pathological and ultrastructural analysis of a case in the head-neck region and a literature review]

BACKGROUND: A high concentration of arachidonic acid in maternal erythrocytes and trophoblast could have a role in pre term deliveries. AIM: To study the fatty acid composition of long chain fatty acids from erythrocytes of mothers who gave birth to pre term and full term infants. PATIENTS AND METHODS: Thirty three healthy women that gave birth to healthy newborns in a public hospital were studied. Twenty two had pre term (34 weeks) and 11 full term (40 weeks) deliveries. The fatty acid profile of phospholipids isolated from erythrocytes of these women was analyzed by gas-liquid chromatography. RESULTS: Compared to women giving birth to full term infants, phospholipids of women giving birth to pre term infants had a higher content of arachidonic acid (20:4 omega 6) and all the species of omega 6 fatty acids. They also had a lower concentration of palmitic and eicosapentanoic (20: 5 omega 3) acids and thus a higher arachidonic acid/eicosapentanoic acid ratio. CONCLUSIONS: A high arachidonic acid content in phospholipids of erythrocytes could be a risk factor or predictive marker for pre term deliveries.     

Yersinia enterocolitica septicemia with septic arthritis

We report a case of Yersinia enterocolitica septicemia with septic arthritis. Gentamicin administration controlled the septicemia but failed to eradicate the organisms in the joint, in spite of a synovial fluid level four times its minimal inhibitory concentration after four days of therapy. Development of azotemia necessitated change of antibiotic therapy to chloramphenicol, which eradicated the infection. While Y enterocolitica infection in the United States is uncommon, it must be added to the list of organisms causing suppurative arthritis and septicemia in susceptible hosts. Septic arthritis must be distinguished from the much more common reactive theumatic polyarthritis associated with Y enterocolimica infection, for which antibiotic therapy is neither needed nor helpful.     

Monohexosylceramides of larval and adult forms of the tapeworm, Spirometra erinacei

The occurrence of glycosphingolipids with unique carbohydrate structures in different species of cestode, Platyhelminth, which had been shown previously, prompted us to study the molecular species of the monohexosylceramides (cerebrosides) in the pseudophyllidean cestode, Spirometra erinacei. The purpose of the study was to obtain a basis for future investigations of the physiological role of glycolipids in parasitism. Cerebrosides were isolated from S. erinacei at two growth stages, i.e., from the larval form (plerocercoid) and from the adult tapeworms (intestinal form). The cerebrosides were separated into four subfractions by silica gel column chromatography, and their constituents were analyzed by gas-liquid chromatography, gas chromatography/mass spectrometry, and high-performance liquid chromatography/mass spectrometry. The hexoses of the cerebrosides consisted primarily of galactose in both growth stages, while only a small amount of glucose was detected. The ceramides were composed of sphinganine (d18:0) and phytosphingosine (t18:0) as sphingoid bases, and of nonhydroxy fatty acids ranging from C16 to C30 and hydroxy stearic acid (18h:0). The cerebrosides of adult tapeworms contained more 18h:0 than those of plerocercoids. The combination of hexoses and ceramides in the cerebroside molecules was slightly different in the two growth stages: the glucocerebrosides of plerocercoids contained only d18:0-nonhydroxy fatty acids in their ceramide moieties, whereas those of adult tapeworms contained varying ceramide moieties. Our data indicate that the molecular species of glycolipids present were essentially homeostatic throughout growth in spite of the entirely different environmental conditions, although there were slight differences in the hexose distribution in the two growth stages.