Osmolal and anion gaps after acute self-poisoning with agricultural formulations of the organophosphorus insecticides profenofos and diazinon : A pilot study

Self-poisoning with organophosphorus (OP) insecticides is an important means of global self-harm. The insecticides are formulated with solvents that may also contribute to toxicity. We set up a study to detect changes in osmolal and anion gaps following ingestion of OP insecticides. We recruited consecutive patients admitted to a Teaching Hospital, Sri Lanka, with a history of OP self-poisoning. The osmolal and anion gaps were calculated on admission and at 4, 24 and 72 h post-ingestion together with ethanol concentration. Forty-nine patients were recruited (28 profenofos, 10 diazinon, one coumaphos, one chlorpyrifos, one phenthoate and eight unknown OP). Only modest increases in osmolal and anion gaps were noted. Small rises in osmolal gap above the upper limit of normal were noted in 16/49 (32.7%) of all cases, 9/28 (32.1%) profenofos cases and 4/10 (40.0%) diazinon cases. The anion gap was raised in 24/49 (49.0%) of all cases, 15/28 (53.6%) profenofos cases and 5/10 (50.0%) diazinon cases. We observed a trend for a fall in osmolal gap during the first 24 h, followed by an increase up to 72 h. There was no correlation between the anion gap and serum lactate concentration, indicating that a lactic acidosis was not responsible for the anion gap. Formate, which could have explained the increased gap, was not detected in any of the samples; ketoacids (beta-hydroxybutyrate and acetoacetate) were not measured. This pilot study found that profenofos and diazinon poisoning caused only modest increases in the osmolal and anion gaps in a minority of cases.Peer reviewe

Osteoporosis and skeletal dysplasia caused by pathogenic variants in SGMS2

Mechanisms leading to osteoporosis are incompletely understood. Genetic disorders with skeletal fragility provide insight into metabolic pathways contributing to bone strength. We evaluated 6 families with rare skeletal phenotypes and osteoporosis by next-generation sequencing. In all the families, we identified a heterozygous variant in SGMS2, a gene prominently expressed in cortical bone and encoding the plasma membrane-resident sphingomyelin synthase SMS2. Four unrelated families shared the same nonsense variant, c.148C>T (p.Arg50*), whereas the other families had a missense variant, c.185T>G (p.IIe62Ser) or c.191T>G (p.Met64Arg). Subjects with p.Arg50* presented with childhood-onset osteoporosis with or without cranial sclerosis. Patients with p.IIe62Ser or p.Met64Arg had a more severe presentation, with neonatal fractures, severe short stature, and spondylometaphyseal dysplasial Several subjects had experienced peripheral facial nerve palsy or other neurological manifestations. Bone biopsies showed markedly altered bone material characteristics, including defective bone mineralization. Osteoclast formation and function in vitro was normal. While the p.Arg50* mutation yielded a catalytically inactive enzyme, p.IIe62Ser and p.Met64Arg each enhanced the rate of de novo sphingomyelin production by blocking export of a functional enzyme from the endoplasmic reticulum. SGMS2 pathogenic variants underlie a spectrum of skeletal conditions, ranging from isolated osteoporosis to complex skeletal dysplasia, suggesting a critical role for plasma membrane-bound sphingomyelin metabolism in skeletal homeostasis.Peer reviewe

Glycolipid transfer protein expression is affected by glycosphingolipid synthesis.

Members of the glycolipid transfer protein superfamily (GLTP) are found from animals and fungi to plants and red micro-alga. Eukaryotes that encode the glucosylceramide synthase responsible for the synthesis of glucosylceramide, the precursor for most glycosphingolipids, also produce GLTPs. Cells that does not synthesize glucosylceramide neither express GLTPs. Based on this genetic relationship there must be a strong correlation between the synthesis of glucosylceramide and GLTPs. To regulate the levels of glycolipids we have used inhibitors of intracellular trafficking, glycosphingolipid synthesis and degradation, and small interfering RNA to down-regulate the activity of glucosylceramide synthase activity. We found that GLTP expression, both at the mRNA and protein levels, is elevated in cells that accumulate glucosylceramide. Monensin and brefeldin A block intracellular vesicular transport mechanisms. Brefeldin A treatment leads to accumulation of newly synthesized glucosylceramide, galactosylceramide and lactosylceramide in a fused endoplasmic reticulum-Golgi complex. On the other hand, inhibiting glycosphingolipid degradation with conduritol-B-epoxide, that generates glucosylceramide accumulation in the lysosomes, did not affect the levels of GLTP. However, glycosphingolipid synthesis inhibitors like PDMP, NB-DNJ and myriocin, all decreased glucosylceramide and GLTP below normal levels. We also found that an 80% loss of glucosylceramide due to glucosylceramide synthase knockdown resulted in a significant reduction in the expression of GLTP. We show here that interfering with membrane trafficking events and simple neutral glycosphingolipid synthesis will affect the expression of GLTP. We postulate that a change in the glucosylceramide balance causes a response in the GLTP expression, and put forward that GLTP might play a role in lipid directing and sensing of glucosylceramide at the ER-Golgi interface

Correction: Glycolipid Transfer Protein Expression Is Affected by Glycosphingolipid Synthesis.

[This corrects the article DOI: 10.1371/journal.pone.0070283.]

Changes in the mass of GlcCer, GalCer and LacCer.

<p>HSF cells were treated with BFA or monensin for 24 h and visualized with orcinol-sulphuric acid on a high performance TLC silica plate. OH-GalCer, hydroxylated GalCer. The representative TLC plate shown here was chosen from one of three independent experiments with similar results.</p

GlcCer, GalCer and Cer and GLTP mRNA levels in HSF cells co-treated with BFA/monensin and different GlcCer synthesis inhibitors.

<p><b>A)</b>³H-sphinganine incorporation and GLTP mRNA levels (filled circles) in HSF cells treated with either BFA (0.01 µg/ml) as well as HSF cells co-treated with BFA in addition to PDMP (50 µM), NB-DNJ (250 µM) and myriocin (25 µM). <b>B)</b>³H-sphinganine incorporation and GLTP mRNA levels (filled circles) in HSF cells treated with either monensin (5 µg/ml) as well as HSF cells co-treated with monensin in addition to PDMP (50 µM), NB-DNJ (250 µM) and myriocin (25 µM). HSF cells treated with myriocin were labeled with ³H-palmitic acid. The results are expressed as means +/− SD of three independent experiments. Two asterisks (**), p<0.01 and three asterisks (***), p<0.005 indicate the statistical significance compared to the controls.</p

Effects of BFA and monensin on GlcCerS, GalCerS and LacCerS mRNA expression levels.

<p>qPCR analysis of the expression levels in cells treated with <b>A)</b> BFA (0.01 µg/ml) and <b>B)</b> monensin (5 µg/ml) for 0, 6, 12 and 24 hours. The qPCR results are expressed as means +/− SD of three independent experiments. <b>C)</b> Scheme of the enzymes in the sphingolipid metabolism analyzed in this study, <i>GalCerS</i> (galactosylceramide synthase) <i>GlcCerS</i> (glucosylceramide synthase) and <i>LacCerS</i> (lactosylceramide synthase).</p

Effect of CBE treatment on GLTP mRNA and protein levels.

<p>HSF cells were treated with CBE (250 µM) for 5 days. <b>A)</b> Simple sphingolipid levels were determined by ³H-sphinganine incorporation and TLC analysis. Incorporation of ³H-sphinganine into GlcCer, GalCer, LacCer, SM and ceramide in untreated controls compared to CBE treated cells. GlcCer (***) levels in CBE treated cells are significantly higher than their controls (p<0.005). <b>B)</b> The GLTP mRNA expression was determined by qPCR in CBE treated HSF cells. Results are expressed as means +/− SD of three independent qPCR experiments. <b>C)</b> The total lipid mass of GlcCer, GalCer and LacCer as visualized by orcinol-sulphuric acid on a HPTLC plate, as well as lipid band intensities semi-quantified using ImageJ software and normalized to the controls. GlcCer (**) levels in CBE treated cells are significantly higher than their controls (p<0.01). <b>D)</b> Western blot of cells treated as described above, C = untreated controls, CBE = 5 day CBE treatment (250 µM). β-Actin was used as a loading control.</p

Expression of GLTP in GlcCerS knockdown HSF cells.

<p>The GLTP levels were analyzed with both qPCR and Western blot in HSF cells with 80% down-regulated expression of the GlcCerS gene. The GLTP gene level was normalized to the level in mock-transfected HSF cells, and β-actin was used as a loading control in the GLTP protein expression analysis.</p

GLTP expression, GlcCer, Galcer, LacCer, ceramide and sphingomyelin synthesis in HSF cells as a function of BFA or monensin treatment.

<p><b>A)</b> HFS cells were treated with BFA (left panel) or monensin (right panel) with increasing concentrations for 24 hours. The GLTP mRNA expression levels were analyzed using qPCR and corrected to an 18S rRNA internal control. <b>B)</b> qPCR analysis of GLTP expression (filled circles) and sphingolipid levels in HSF cells treated with BFA (0.01 µg/ml, left panel) or monensin (5 µg/ml, right panel) for 6, 12 and 24 hours, ³H-sphinganine incorporation into the sphingolipids was analyzed using TLC. qPCR results are expressed as means +/− SD of at least three independent experiments. The data for the incorporation of the radiolabeled ³H-sphinganine are from at least three different experiments, and the results are normalized to the controls. Two asterisks (**), p<0.01 and three asterisks (***), p<0.005 indicate the statistical significance compared to the controls. <b>C)</b> Western blot analysis of GLTP levels in HSF cells treated with BFA (0.01 µg/ml) or monensin (5 µg/ml) for 24 hours. C = untreated control, B = BFA treatment, M = monensin treatment. β-Actin was used as a loading control. The representative blot shown here was chosen from one of three independent experiments with similar results.</p