UK Reference Intervals for Parathyroid Hormone Using Abbott Methods.

Background: Diagnosis of hyperparathyroidism requires measurement of parathyroid hormone (PTH) in the context of the plasma calcium and other factors, such as vitamin D status and renal function. Accurate classification depends upon an appropriate population reference interval. We examined local population plasma PTH reference intervals at four different UK sites using a common platform. Methods: Plasma PTH results were extracted from laboratory information systems at four different UK sites, all using the Abbott Architect i2000 method. We included only people with normal adjusted serum calcium, magnesium, vitamin D, and renal function. Following outlier rejection lower and upper reference limits were derived. Results: An overall reference interval for plasma PTH of 3.0-13.7 pmol/L was observed using a non-parametric approach compared to 2.9-14.1 pmol/L using a parametric approach, notably higher than the manufacturer's representative range of 1.6-7.2 pmol/L. We also noted statistically significant differences (p < 0.00001) between some sites with upper limits ranging from 11.5 to 15.8 pmol/L which may be due to different population characteristics of each group. Conclusion: Locally derived reference intervals may be beneficial for UK populations and revised upper thresholds are necessary when using the Abbott PTH method to avoid inappropriate classification of patients as having hyperparathyroidism.

Branding Practices on Four Dairies in Kantale, Sri Lanka.

Hot-iron branding is illegal in Sri Lanka, but is still commonly used to identify dairy herds in extensive farming systems, which are primarily located in the country's Dry Zone. Despite the negative welfare implications of this practice, there is no written documentation of branding in this region. We observed branding on four smallholder farms in Kantale, Eastern Province to understand the welfare implications associated with the procedure and challenges limiting the uptake of more welfare-friendly alternatives, such as ear tagging. Areas of welfare concern included the duration of restraint, the size and location of the brand, and the absence of pain relief. Animals were restrained with rope for an average duration of 12 min (range 8⁻17 min). Farmers used multiple running irons to mark their initials and, in some cases, their address, with the largest brands extending across the ribs and hip. Three farmers applied coconut or neem oil topically to the brand after performing the procedure. No analgesics were given before or after branding. Farmers reported that poor ear tag retention in extensive systems and theft were the main factors impeding the uptake of alternative forms of identification. Branding is also practiced as part of traditional medicine in some cases. Given the clear evidence that hot-iron branding impairs animal welfare and there is no evidence that this can be improved, alternative identification methods are needed, both in Sri Lanka, as well as in other countries engaging in this practice.

Use of FGF-21 as a Biomarker of Mitochondrial Disease in Clinical Practice.

Recent work has suggested that fibroblast growth factor-21 (FGF-21) is a useful biomarker of mitochondrial disease (MD). We routinely measured FGF-21 levels on patients who were investigated at our centre for MD and evaluated its diagnostic performance based on detailed genetic and other laboratory findings. Patients' FGF-21 results were assessed by the use of age-adjusted z-scores based on normalised FGF-21 values from a healthy population. One hundred and fifty five patients were investigated. One hundred and four of these patients had molecular evidence for MD, 27 were deemed to have disorders other than MD (non-MD), and 24 had possible MD. Patients with defects in mitochondrial DNA (mtDNA) maintenance (n = 32) and mtDNA rearrangements (n = 17) had the highest median FGF-21 among the MD group. Other MD patients harbouring mtDNA point mutations (n = 40) or mutations in other autosomal genes (n = 7) and those with partially characterised MD had lower FGF-21 levels. The area under the receiver operating characteristic curve for distinguishing MD from non-MD patients was 0.69. No correlation between FGF-21 and creatinine, creatine kinase, or cardio-skeletal myopathy score was found. FGF-21 was significantly associated with plasma lactate and ocular myopathy. Although FGF-21 was found to have a low sensitivity for detecting MD, at a z-score of 2.8, its specificity was above 90%. We suggest that a high serum concentration of FGF-21 would be clinically useful in MD, especially in adult patients with chronic progressive external ophthalmoplegia, and may enable bypassing muscle biopsy and directly opting for genetic analysis. Availability of its assay has thus modified our diagnostic pathway.

Regional protein levels of cytosolic phospholipase A2 and cyclooxygenase-2 in Rhesus monkey brain as a function of age.

Limited evidence suggests that brain cytosolic phospholipase A(2) (cPLA(2)), which selectively releases arachidonic acid (AA) from membrane phospholipids, and cyclooxygenase-2 (COX-2), the rate-limiting enzyme for AA metabolism to prostanoids, change as a function of normal aging. In this study, we examined the protein levels of cPLA(2) and COX-2 enzymes in hippocampus, frontal pole and cerebellum from young (2-5 years old), middle-aged (8-11 years old) and old (23 years old) male and female Rhesus monkeys. In the cerebellum, cPLA(2) protein level was higher in the young brain as compared to levels seen at both middle-aged and old. Similarly, in the frontal pole, the young brain showed a higher level of COX-2 protein as compared to the levels seen at both older ages. For both, once an animal reached 8-11 years of age the levels appeared to remain relatively constant over the next decade. Immunohistochemistry of COX-2 protein within the brain demonstrated no significant change in the localization to neurons within the frontal pole. Qualitatively, a greater number of neurons were positively stained for COX-2 in the young brain than in the aged brain. Based on the previous reports of localization of cPLA(2) and COX-2 at post-synaptic sites in neurons results from the current study suggest that the elevated protein levels of the two enzymes seen in the younger brain is related to the greater potential for synaptic plasticity across multiple neurons as a function of age and that cPLA(2) and COX-2 may be considered as post-synaptic markers.

The effect of chronic lithium on arachidonic acid release and metabolism in rat brain does not involve secretory phospholipase A2 or lipoxygenase/cytochrome P450 pathways.

The mood-stabilizer lithium, when chronically administered to rats at therapeutic concentrations, has been shown to downregulate brain arachidonic acid (AA) turnover and total phospholipase A2 (PLA2) activity, as well as protein and mRNA levels of cytosolic cPLA2. These effects are accompanied by a decrease in cyclooxygenase (COX)-2 protein level, COX activity, and brain prostaglandin E2 (PGE2) concentration. The involvement of Ca2+-dependent secretory PLA2 (sPLA2) in the mechanism of action of lithium has not been investigated. The purpose of this study was to examine, whether the effect of lithium is selectively directed to cPLA2 or it also affects sPLA2 protein and enzyme activity and whether other AA metabolizing enzymes (5-lipoxygenase and cytochrome P450 epoxygenase) were also altered. Furthermore, to determine if the reduction of brain PGE2 concentration was due only to downregulation of COX-2 protein or if it also involves the terminal PGE synthase, we determined brain microsomal PGE synthase protein level. Male Fischer-344 rats were fed lithium chloride for 6 weeks, whereas, control rats were fed lithium-free chow under parallel conditions. We found that chronic lithium did not significantly change sPLA2 activity or protein level. 5-Lipoxygenase and cytochrome P450 epoxygenase protein levels were unchanged, as were levels of the terminal PGE synthase. These results indicate that the effect of lithium selectively involves the cPLA2/COX-2 pathway, which might be responsible for the therapeutic effect in bipolar disorder.

Lithium chloride, administered chronically to rats, does not affect the fractional phosphorylation of brain cytosolic phospholipase A2, while reducing its net protein level.

Lithium, used to treat bipolar disorder, has been reported to decrease rat brain mRNA and protein levels of cytosolic phospholipase A(2) (cPLA(2)), an enzyme that selectively hydrolyzes arachidonic acid from the stereospecifically numbered (sn)-2 position of membrane phospholipids, and to decrease PLA(2) activity. cPLA(2) can be activated by being phosphorylated at its Ser-228, Ser-505, and Ser-727 sites. In this study, we show that the percent phosphorylated cPLA(2) protein in rat brain is unaffected by lithium. Male Fischer-344 rats were fed lithium chloride for 6 weeks, so as to produce a therapeutically equivalent brain lithium concentration; control rats were fed lithium-free chow under parallel conditions. cPLA(2) was immunoprecipitated from brain homogenate and phosphorylated cPLA(2) protein was quantified using an anti-phosphoserine antibody, and compared to net cPLA(2) protein. The mean ratio of phosphorylated/total cPLA(2) was not changed significantly in the lithium-treated compared to the control group. Thus, decreased brain PLA(2) enzyme activity caused by chronic lithium is likely a consequence only of lithium's downregulation of cPLA(2) transcription.

A new histochemical approach for studying sperm cell surfaces.

This study describes a novel rapid histochemical method to survey surface properties of sperm in a model system, the sea urchin. Surface properties of live and fixed sea urchin sperm of 2 species (Strongylocentrotus purpuratus and Lytechinus pictus) in seawater (live and fixed) and distilled water (fixed) were surveyed by assessing the ability of the sperm to bind to agarose beads derivatized with over 100 lectins and other proteins, carbohydrates, amino acids, nucleotides and other molecules. The results indicate that very little cell-bead binding occurs in seawater, whereas a great deal of binding occurs in distilled water, suggesting that ions inhibit cell-bead interaction. Whereas sperm of both species bound to many of the same beads, there were substantial differences in binding of sperm of each species to some beads, and there were differences in sperm-bead binding at different times of the year. The results suggest that the bead-binding assay enables the identification of species-specific and seasonally variable cell- surface properties. The large number of probes (bead types) available in the bead assay increases the likelihood of identifying new cell surface markers whose functional significance can then be the subject of further investigation. This assay offers an entirely new approach to examine surfaces of mammalian sperm in studies that could lead to identification of receptors involved in sperm-egg interaction, that may be candidates for the development of new contraceptive strategies.

Analysis of surface properties of fixed and live cells using derivatized agarose beads.

A novel assay has been developed for the histochemical characterization of surface properties of cells based on their adhesion to agarose beads derivatized with more than 100 types of molecules, including sugars, lectins and other proteins, and amino acids. The assay simply involves mixing small quantities of washed cells and beads in droplets on glass microscope slides and determining to which beads various cell types adhere. Distilled water was found to be the best medium for this assay because added ions or molecules in other media inhibit adhesion in some cases. Many cells, however, cannot tolerate distilled water. Here we show that cells fixed with either of two fixatives (1% formaldehyde or Prefer fixative) displayed similar bead-binding properties as did live cells. Specificity of cell-bead binding was tested by including specific free molecules in the test suspensions in hapten-type inhibition experiments. If a hapten compound inhibited live-cell adhesion to a specific bead, it also inhibited fixed-cell adhesion to a specific bead. The results of these experiments suggest that fixed cells display authentic surface properties, opening the door for the use of this assay with many cell types that cannot tolerate distilled water.

The charged milieu: a major player in fertilization reactions.

In previous studies, we have found that negatively charged, but not uncharged, amino acids and sugars block sea urchin fertilization. These studies were developed from modeling work in non-living systems using derivatized agarose beads that suggested that charge-charge bonding may control at least some adhesive interactions. In the present study, the effects of positively charged, negatively charged and uncharged molecules were examined in the sea urchin sperm-egg system in over 300 individual trials. The results indicate that depending on the specific molecules utilized, both sperm and egg are exquisitely sensitive to charged but not uncharged molecules and to pH changes in sea water caused by some of the charged molecules. It is shown that egg activation, as well as sperm motility and sperm-egg interactions, can be affected by charged molecules. One compound, fructose-1-phosphate blocked fertilization in S. purpuratus sea urchins but not in Lytechinus pictus sea urchins. These findings indicate that charge alone cannot explain all the results. In this case, the presence of a ketone instead of an aldehyde group indicates that species-specific components may control fertilization reactions. The present study is a comprehensive survey of the effects of charge, pH and molecular structure on the fertilization activation continuum in a model system of sea urchins.

Analysis of surface properties of human cancer cells using derivatized beads.

Standard histochemical analysis of cells and tissues generally involves procedures that utilize a relatively small number of probes such as dyes, and generally requires hours or days to process. Our laboratory has developed a novel method for histochemical surveys of cell surface properties that utilizes a large number of probes (derivatized agarose beads) and takes seconds or minutes to accomplish. In this study, 4 human cell lines (CCL-255 (LS123) human colon cancer cells that are non-tumorigenic in nude mice; CRL-1459 (CCD-18CO) human colon endothelial cells that are non-malignant; CCL-220 (COLO 320DM) human colon cancer cells that are tumorigenic in nude mice; and HTB-171 (NCI H446) human lung carcinoma cells) were tested for their ability to bind to agarose beads derivatized with 51 different molecules. There were statistically significant differences in binding of the 4 cell types to all of the 51 types of beads, but 15 types of beads showed dramatic differences in binding to one or more of the 4 cell types. For example, only HTB-171 (NCI H446) bound to p-aminophenyl-beta-D-glucopyranoside-derivatized beads and only CCL-220 (COLO 320DM) bound to L-tyrosine-derivatized beads. The specificity of cell-bead binding was examined by performing assays in the presence or absence of exogenously added compounds in hapten-type of inhibition experiments. This assay, that utilizes large numbers of novel probes, may help in the development of new libraries of surface properties of specific cell types, with differing degrees of malignancy, that at this time could not be developed by using other available technologies.

O-linked-N-acetylglucosamine cycling and insulin signaling are required for the glucose stress response in Caenorhabditis elegans.

In a variety of organisms, including worms, flies, and mammals, glucose homeostasis is maintained by insulin-like signaling in a robust network of opposing and complementary signaling pathways. The hexosamine signaling pathway, terminating in O-linked-N-acetylglucosamine (O-GlcNAc) cycling, is a key sensor of nutrient status and has been genetically linked to the regulation of insulin signaling in Caenorhabditis elegans. Here we demonstrate that O-GlcNAc cycling and insulin signaling are both essential components of the C. elegans response to glucose stress. A number of insulin-dependent processes were found to be sensitive to glucose stress, including fertility, reproductive timing, and dauer formation, yet each of these differed in their threshold of sensitivity to glucose excess. Our findings suggest that O-GlcNAc cycling and insulin signaling are both required for a robust and adaptable response to glucose stress, but these two pathways show complex and interdependent roles in the maintenance of glucose-insulin homeostasis.

The expression of brain cyclooxygenase-2 is down-regulated in the cytosolic phospholipase A2 knockout mouse.

We examined brain phospholipase A2 (PLA2) activity and the expression of enzymes metabolizing arachidonic acid (AA) in cytosolic PLA2 knockout () mice to see if other brain PLA2 can compensate for the absence of cPLA2 alpha and if cPLA2 couples with specific downstream enzymes in the eicosanoid biosynthetic pathway. We found that the rate of formation of prostaglandin E2 (PGE2), an index of net cyclooxygenase (COX) activity, was decreased by 62% in the compared with the control mouse brain. The decrease was accompanied by a 50-60% decrease in mRNA and protein levels of COX-2, but no change in these levels in COX-1 or in PGE synthase. Brain 5-lipoxygenase (5-LO) and cytochrome P450 epoxygenase (cyp2C11) protein levels were also unaltered. Total and Ca2+-dependent PLA2 activities did not differ significantly between and control mice, and protein levels of type VI iPLA2 and type V sPLA2, normalized to actin, were unchanged. These results show that type V sPLA2 and type VI iPLA2 do not compensate for the loss of brain cPLA2 alpha, and that this loss has significant downstream effects on COX-2 expression and PGE2 formation, sparing other AA oxidative enzymes. This suggests that cPLA2 is critical for COX-2-derived eicosanoid production in mouse brain.

Prostaglandin E2 and microsomal prostaglandin E synthase-2 expression are decreased in the cyclooxygenase-2-deficient mouse brain despite compensatory induction of cyclooxygenase-1 and Ca2+-dependent phospholipase A2.

We previously demonstrated that brain cyclooxygenase (COX)-2 mRNA and protein levels, and prostaglandin E2 (PGE2) level, are down-regulated in cytosolic phospholipase A2 (cPLA2) -deficient mice. To further investigate the interaction between upstream and downstream enzymes involved in brain prostaglandin synthesis, we examined expression and activity of COX-1, of different PLA2 enzymes and of prostaglandin E synthase (PGES) enzymes in COX-2(-/-) mice. We found that the PGE2 level was decreased by 51.5% in the COX-2(-/-) mice brains, indicating a significant role of COX-2 in brain formation of PGE2. However, when we supplied exogenous arachidonic acid (AA) to brain homogenates, COX activity was increased in the COX-2(-/-) mice, suggesting a compensatory activation of COX-1 and an intracellular compartmentalization of the COX isozymes. Consistent with COX-1 increased activity, brain expression of COX-1 protein and mRNA also was increased. Activity and expression of cPLA2 and secretory PLA2 (sPLA2) enzymes, supplying AA to COX, were significantly increased. Also, the PGE2 biosynthetic pathway downstream from COX-2 was affected in the COX-2(-/-) mice, as decreased expression of microsomal prostaglandin E synthase-2 (mPGES-2), but not mPGES-1 or cytosolic PGES, was observed. Overall, the data suggest that compensatory mechanisms exist in COX-2(-/-) mice and that mPGES-2 is functionally coupled with COX-2.

Valproic acid down-regulates the conversion of arachidonic acid to eicosanoids via cyclooxygenase-1 and -2 in rat brain.

Sodium valproate, a mood stabilizer, when chronically administered to rats (200 mg/kg i.p. daily for 30 days) significantly reduced the brain protein levels of cyclooxygenase (COX)-1 and COX-2, without altering the mRNA levels of these enzymes. COX activity was decreased, as were the brain concentrations of 11-dehydrothromboxane B2 and prostaglandin E2 (PGE2), metabolites of arachidonic acid (AA) produced via COX. In contrast, the brain protein level of 5-lipoxygenase and the concentration of its AA metabolite leukotriene B4 were unchanged. In view of published evidence that lithium chloride administered chronically to rats, like chronic valproate, reduces AA turnover within brain phospholipids, and that lithium post-transcriptionally down-regulates COX-2 but not COX-1 protein level and enzyme activity, these observations suggest that mood stabilizers generally modulate the release and recycling of AA within brain phospholipids, and the conversion of AA via COX-2 to PGE2 and related eicosanoids. If targeting this part of the 'AA cascade' accounts for their therapeutic action, non-steroidal anti-inflammatory drugs or selective COX-2 inhibitors might prove effective in bipolar disorder.

Rat brain arachidonic acid metabolism is increased by a 6-day intracerebral ventricular infusion of bacterial lipopolysaccharide.

In a rat model of acute neuroinflammation, produced by a 6-day intracerebral ventricular infusion of bacterial lipopolysaccharide (LPS), we measured brain activities and protein levels of three phospholipases A2 (PLA2) and of cyclo-oxygenase-1 and -2, and quantified other aspects of brain phospholipid and fatty acid metabolism. The 6-day intracerebral ventricular infusion increased lectin-reactive microglia in the cerebral ventricles, pia mater, and the glial membrane of the cortex and resulted in morphological changes of glial fibrillary acidic protein (GFAP)-positive astrocytes in the cortical mantel and areas surrounding the cerebral ventricles. LPS infusion increased brain cytosolic and secretory PLA2 activities by 71% and 47%, respectively, as well as the brain concentrations of non-esterified linoleic and arachidonic acids, and of prostaglandins E2 and D2. LPS infusion also increased rates of incorporation and turnover of arachidonic acid in phosphatidylethanolamine, plasmenylethanolamine, phosphatidylcholine, and plasmenylcholine by 1.5- to 2.8-fold, without changing these rates in phosphatidylserine or phosphatidylinositol. These observations suggest that selective alterations in brain arachidonic acid metabolism involving cytosolic and secretory PLA2 contribute to early pathology in neuroinflammation.