A patient with hyperglycaemia and normal HbA Ic due to impaired glycation.

A diabetic Caucasian woman presented with discrepantly low HbA Ic values compared with her glycaemia. High-performance liquid chromatography (HPLC) analysis disclosed 80% HbA and 20% HbI Philadelphia (I6alpha2 lys --> glut). The calculated glycosylation gap from the fructosamine level was 1.2%. The haemoglobin alpha/beta glycation ratios, as measured by electron spray ionisation mass spectroscopy (ESI-MS), for the patient and her three children also carrying the mutation were decreased by values of 0.56 and 0.51, 0.50 and 0.49, respectively (reference value 0.66).

Endotoxin binding to platelets in blood from patients with a sepsis syndrome.

Endotoxin, the lipopolysaccharide cell wall constituent of Gram-negative bacteria, produces symptoms of the Gram-negative sepsis syndrome. By measuring endotoxin in blood from septic patients it may be possible to select a subpopulation of patients in which mortality can be prevented by treatment with anti-endotoxin antibodies. We evaluated the performance of an endotoxin-free blood-collection tube. Within-run and between-run CVs of our endotoxin assay were 4-18% and 8-20%, respectively. In endotoxin-positive samples (LPS > or = 6 ng/L), the concentration of endotoxin in platelet-rich plasma was significantly higher (P < 0.001) than in platelet-poor plasma. Apparent binding of endotoxin to platelets ranged from 0% to 92%. The correlation between the apparent percentage binding of LPS to platelets and the platelet count in platelet-rich plasma is linear and positive, but LPS is not bound solely to platelets. We conclude that endotoxin must be measured in platelet-rich plasma.

Laboratory and clinical evaluation of a chromogenic endotoxin assay for horses with acute intestinal disorders.

In this study the laboratory and clinical performance of a chromogenic endotoxin assay for equine plasma was evaluated. The assay was sensitive (detection limit 3 ng LPS/L plasma), reproducible (within and between-assay CV at 50 ng LPS/L E. coli O111:B4 LPS standard addition was 5% and 7.5%, respectively), and not substantially affected by enhancement or inhibition phenomena (recovery of an in vitro spike was 75-125% in 80% of the samples). LPS added to whole blood was rapidly inactivated upon incubation at 37 degrees C but not at 0 degrees C. A recently developed blood collection tube for LPS testing was found suitable, i.e. LPS-free and providing non-contaminated samples. In 48 horses suffering from acute abdominal diseases requiring surgical treatment, LPS levels were significantly higher in platelet-rich plasma (PRP) than in platelet-poor plasma (PPP), and the proportional difference was related to the PRP platelet count (r = 0.52, p < 0.001, mean difference 48%, range 8-77%). LPS levels were also significantly higher in horses that died or were euthanized than in surviving horses (mean 16.5 and 7.1 ng/L PRP, respectively, p < 0.05). We conclude that LPS can be measured in equine plasma with picogram sensitivity and recommend the use of PRP instead of PPP for clinical LPS testing. For clinical use a decision limit for endotoxaemia of 5 ng LPS/L PRP appeared to be inadequate. Analysis at a higher cut-off level for endotoxaemia and the evaluation of clinical, pathological, and laboratory parameters would be more meaningful.

Analytical performance of three commercially available nephelometers compared for quantifying proteins in serum and cerebrospinal fluid.

We investigated the analytical performance and imprecision of three commercially available nephelometers for the quantification of various proteins in pooled serum and cerebrospinal fluid. Albumin, transferrin, IgG, IgA, and IgM in serum were determined nephelometrically (BNA, Behring; Array, Beckman) and turbidimetrically (Cobas Bio, Hoffmann-La Roche). All these proteins in cerebrospinal fluid except transferrin were determined nephelometrically with all three systems. CVs and lower limits of detection were compared for all instruments, and stability of calibration curves was evaluated.

The effect of propylbenzilylcholine mustard on contraction and radioligand binding parameters of muscarinic receptors in guinea pig ileum.

The receptor occupancy-biological effect relationship for muscarinic receptors in guinea pig ileal smooth muscle has been studied by comparison of radioligand binding and contractile response. Muscarinic receptors in homogenates of ileal smooth muscle were labeled with [3H]-1-Quinuclidinyl benzilate. Treatment with propylbenzilylcholine mustard (PrBCM), to inactivate irreversibly muscarinic receptors, caused a large dose dependent rightward shift of the dose-response curve to three agonistic furtrethonium derivatives with a concomitant decrease in maximal response. Using those data, the fraction of receptors remaining unoccupied (q-values) and "true affinity constants" (-log KA-values) were calculated. Exposure to 20 or 60 nM PrBCM for 15 minutes resulted in a 39% and a 61% reduction in specific [3H]-1-Quinuclidinyl benzilate binding sites respectively to be compared with a 62% and a 85% decrease expected from calculated q-values. KA-values for the methyl and ethyl derivative agreed well with the dissociation constants for the high affinity agonist sites determined from displacement of [3H-]-1-Quinuclidinyl benzilate. The KA-value for the propylfurtrethonium corresponds to the low affinity agonist dissociation constant. The fraction of receptors in the high affinity agonist state differs considerably for the three furtrethonium derivatives investigated. Neither the fraction of receptors in the high affinity agonist state, nor the ratio of dissociation constants for these states is affected by the alkylation of 85% of the functional muscarinic receptors. The inactivation of components of the effector system by PrBCM seems unlikely.

Muscarinic receptors in rat nasal mucosa are predominantly of the low affinity agonist type.

Specific [3H]l-quinuclidinyl benzilate binding to rat nasal mucosa homogenates occurs to a homogeneous class of binding sites with Kd = 60 +/- 2 10(-12) M and Bmax = 8.1 +/- 2 pmol/g tissue. Binding is stereoselectively inhibited by benzetimide enantiomers. Pirenzepine inhibits [3H]l-quinuclidinyl benzilate binding with low affinity (Ki = 5.0 10(-7) M), classifying the binding sites as muscarinic M2-receptors. Methylfurtrethonium and methacholine inhibit [3H]l-quinuclidinyl benzilate binding following an almost sigmoid curve at high concentrations pointing to the presence of mainly low affinity agonist binding sites.

The metabolic fate of branched-chain amino acids and 2-oxo acids in rat muscle homogenates and diaphragms.

After incubation of muscle preparations with [U-14C]branched-chain amino acids or 2-oxo acids, radioactive metabolites were separated, identified and quantified. Homogenates of rat heart and skeletal muscle incubated with 4-methyl-2-oxopentanoate accumulated isovalerate, 3-hydroxyisovalerate and the corresponding carnitine esters. Incubation with 3-methyl-2-oxobutanoate resulted in the production of isobutyrate, 3-hydroxyisobutyrate and their carnitine esters. Addition of L-carnitine increased the production of the esters. The enzymes 3-methylcrotonyl-CoA carboxylase and 3-hydroxyisobutyric acid dehydrogenase apparently are inactive during incubation of muscle homogenates. With liver homogenates the degradation of both 2-oxo acids was more complete. Rat hemidiaphragms incubated with leucine, valine and isoleucine accumulated the corresponding branched-chain 2-oxo acids, fatty acids and hydroxylated fatty acids. The degradation of valine was markedly limited by the release of these metabolites. Considerable amounts (relatively smaller for valine) of radioactivity were also recovered in CO2 and glutamine and glutamate. Incubations with branched-chain 2-oxo acids gave the same radioactive products, except for glutamine and glutamate. Radioactivity was never found in lactate, pyruvate or alanine. These data indicate that the carbon-chains of amino acids entering the citric acid cycle in muscle, are not used for oxidation or for alanine synthesis, but are converted exclusively to glutamine.