Status on fasting definition for blood sampling in the Nordic countries - time for a harmonized definition.

The preanalytical phase contains a vast number of practices whose variation may influence the results of laboratory testing and should, therefore, be standardized. The Working Group on Preanalytical Phase of the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM WG-PA) has suggested a standardization of venous blood specimen collection (VBSC) requirements for fasting samples including 12 h fasting time and water ad lib in the morning prior to specimen collection. The Nordic Scientific Preanalytical Working Group investigated the fasting definitions used in the Nordic countries. The Internet was assessed for stated fasting definitions of official organizations, larger laboratories, or laboratory groups. Fasting instructions for VBSC generally demanded patients to abstain from alcohol a day prior to, and to abstain from coffee, tea, smoking, and snuff intake in the morning of VBSC. Norway had a national fasting definition. Required fasting times varied from 8 to 14 h. The amount of water allowed in the morning of VBSC varied from ad lib to half a glass of water. The list of analytes, where fasting was required, held 9-15 analytes except for Finland with 65 analytes. Implementation of the EFLM WG-PRE standardization of VBSC requirements for fasting samples would decrease preanalytical variability and be beneficial for medical decisions and patient data comparison. We suggest the laboratories in the Nordic countries to implement the suggested fasting requirements, which are in line with those used when fasting reference intervals were established in the Nordic reference interval project.

Hypopituitarism 3 and 12 months after traumatic brain injury and subarachnoid haemorrhage.

OBJECTIVE: High prevalence of hypopituitarism (HP) has been reported after traumatic brain injury (TBI) and subarachnoid haemorrhage (SAH). The aim of the study was to prospectively evaluate the prevalence and progression of HP in patients after TBI and SAH in Icelandic population. DESIGN: A 12 month prospective single-centre study. METHODS AND PROCEDURES: A total of 27 patients were included, 15 patients with TBI and 12 patients with SAH. Pituitary function was evaluated with baseline hormone measurements and diagnostic tests. An insulin tolerance test was used unless contraindicated, then the GHRH-arginine test and Synachten test were used. RESULTS: At three months, 16.7% (2/12) of the patients had HP after TBI and 33.3% (4/12) after SAH. At 12 months, 21.4% (3/14) of patients had HP after TBI and 9.1% (1/11) after SAH. Gonadotropin deficiency was the most common deficiency at 3 months and GH and gonadotropin deficiency at 12 months. CONCLUSIONS: There is a considerable risk of HP after TBI and reason to study pituitary function further in patients with SAH. We believe that neuroendocrine evaluation is important in these patients. Since recovery commonly occurs 12 months after the event, evaluation should be performed after that time if not clinically indicated earlier.

Structure-activity relationship studies leading to the identification of (2E)-3-[l-[(2,4-dichlorophenyl)methyl]-5-fluoro-3-methyl-lH-indol-7-yl]-N-[(4,5-dichloro-2-thienyl)sulfonyl]-2-propenamide (DG-041), a potent and selective prostanoid EP3 receptor antagonist, as a novel antiplatelet agent that does not prolong bleeding.

The EP(3) receptor on the platelet mediates prostaglandin E(2) potentiation of thrombogenic coagonists including collagen and adenosine diphosphate (ADP). A pharmacophore driven approach led to the identification of diverse peri-substituted heterocycles as potent and selective EP(3) receptor antagonists. A simultaneous chemical optimization and druglike assessment of prioritized molecules converged on a lead compound 50 (DG-041) that displayed favorable in vitro and functional activities as an inhibitor of human platelet aggregation. This agent is currently in human clinical trials for the treatment of atherothrombosis.

Discovery of 4-[(2S)-2-{[4-(4-chlorophenoxy)phenoxy]methyl}-1-pyrrolidinyl]butanoic acid (DG-051) as a novel leukotriene A4 hydrolase inhibitor of leukotriene B4 biosynthesis.

Both in-house human genetic and literature data have converged on the identification of leukotriene 4 hydrolase (LTA(4)H) as a key target for the treatment of cardiovascular disease. We combined fragment-based crystallography screening with an iterative medicinal chemistry effort to optimize inhibitors of LTA(4)H. Ligand efficiency was followed throughout our structure-activity studies. As applied within the context of LTA(4)H inhibitor design, the chemistry team was able to design a potent compound 20 (DG-051) (K(d) = 26 nM) with high aqueous solubility (>30 mg/mL) and high oral bioavailability (>80% across species) that is currently undergoing clinical evaluation for the treatment of myocardial infarction and stroke. The structural biology-chemistry interaction described in this paper provides a sound alternative to conventional screening techniques. This is the first example of a gene-to-clinic paradigm enabled by a fragment-based drug discovery effort.

Antagonists of the EP3 receptor for prostaglandin E2 are novel antiplatelet agents that do not prolong bleeding.

Myocardial infarction and stroke are caused by blood clots forming over a ruptured or denuded atherosclerotic plaque (atherothrombosis). Production of prostaglandin E(2) (PGE(2)) by an inflamed plaque exacerbates atherothrombosis and may limit the effectiveness of current therapeutics. Platelets express multiple G-protein coupled receptors, including receptors for ADP and PGE(2). ADP can mobilize Ca(2+) and through the P(2)Y(12) receptor can inhibit cAMP production, causing platelet activation and aggregation. Clopidogrel (Plavix), a selective P(2)Y(12) antagonist, prevents platelets from clotting but thereby increases the risk of severe or fatal bleeding. The platelet EP(3) receptor for PGE(2), like the P(2)Y(12) receptor, also inhibits cAMP synthesis. However, unlike ADP, facilitation of platelet aggregation via the PGE(2)/EP(3) pathway is dependent on co-agonists that can mobilize Ca(2+). We used a ligand-based design strategy to develop peri-substituted bicylic acylsulfonamides as potent and selective EP(3) antagonists. We show that DG-041, a selective EP(3) antagonist, inhibits PGE(2) facilitation of platelet aggregation in vitro and ex vivo. PGE(2) can resensitize platelets to agonist even when the P(2)Y(12) receptor has been blocked by clopidogrel, and this can be inhibited by DG-041. Unlike clopidogrel, DG-041 does not affect bleeding time in rats, nor is bleeding time further increased when DG-041 is co-administered with clopidogrel. This indicates that EP(3) antagonists potentially have a superior safety profile compared to P(2)Y(12) antagonists and represent a novel class of antiplatelet agents.

Role of bile in pathogenesis of indomethacin-induced enteropathy.

Ingestion of non-steroidal anti-inflammatory drugs (NSAIDs) causes an enteropathy. The pathogenesis involves biochemical initiation of intestinal mucosal damage due to NSAID-induced inhibition of cyclooxygenase and the topical effects of these drugs. These effects lead to increased intestinal permeability and inflammation. Luminal bile acids play a controversial role in the damage produced by these drugs. The aim of this study was to determine the role of bile in producing the enteropathy caused by indomethacin, an NSAID commonly used in toxicity studies. Sprague-Dawley rats were subjected to bile duct ligation. Twenty-four hours later, they were dosed with indomethacin. Intestinal permeability ((51)Cr-EDTA) and inflammation (faecal calprotectin) were measured in the animals at various time periods after the dose. Intestinal permeability was significantly higher in rats 1-6 h after dosing with indomethacin, but not at 24-29 h or day 4, when compared with corresponding values for control animals. Excretion of faecal calprotectin was elevated in the indomethacin-treated rats. The drug-treated animals showed no evidence of ulceration when they were sacrificed 29 h or a week after the dose of indomethacin. Bile acids per se did not affect intestinal permeability or faecal excretion of calprotectin, when given along with indomethacin or its vehicle. We conclude that macroscopic small bowel damage does not occur with indomethacin if bile is excluded, despite the induction of permeability and inflammation. This study highlights the importance of luminal factors, such as bile, in producing indomethacin-induced ulceration in the rat small intestine.

Use of surrogate markers of inflammation and Rome criteria to distinguish organic from nonorganic intestinal disease.

BACKGROUND & AIMS: Differentiating symptoms of irritable bowel syndrome (IBS) from those of organic intestinal disease is a familiar problem for physicians. The aim of this study was to assess the sensitivity, specificity, and odds ratios (ORs) of fecal calprotectin, small intestinal permeability, Rome I criteria, and laboratory markers of inflammation (erythrocyte sedimentation rate [ESR], C-reactive protein [CRP], blood count) in distinguishing organic from nonorganic intestinal disease. METHODS: A total of 602 new referrals to a gastroenterology clinic who had symptoms suggestive of IBS or organic intestinal disease were studied for these parameters. All patients underwent invasive imaging (barium/endoscopic examination) and other investigations as appropriate, with physicians blinded to the results of fecal calprotectin and intestinal permeability. RESULTS: A total of 263 patients were diagnosed with organic disease and 339 with IBS. At 10 mg/L, the sensitivity and specificity of calprotectin for organic disease were 89% and 79%, respectively, and that of intestinal permeability for small intestinal disease were 63% and 87%, respectively. Sensitivity of positive Rome criteria for IBS was 85% with a specificity of 71%. An abnormal calprotectin test had an OR for disease of 27.8 (95% confidence interval [CI], 17.6-43.7; P < 0.0001) compared with ORs of 4.2 (95% CI, 2.9-6.1; P < 0.0001) and 3.2 (95% CI, 2.2-4.6; P < 0.0001) for elevated CRP and ESR values. An abnormal permeability test gave an OR of 8.9 (95% CI, 5.8-14.0; P < 0.0001) for small intestinal disease. The OR for IBS with positive Rome criteria was 13.3 (95% CI, 8.9-20.0). CONCLUSIONS: Fecal calprotectin, intestinal permeability, and positive Rome I criteria provide a safe and noninvasive means of helping differentiate between patients with organic and nonorganic intestinal disease.

[Effect of Pentavac and MMR vaccination on the intestine.].

OBJECTIVE: The safety of infant vaccination has been questioned in recent years. In particular it has been suggested that the measles, mumps and rubella (MMR) vaccination leads to brain damage manifesting as autism consequent to the development of an "enterocolitis" in the immediate post-vaccination period. AIM: To assess if MMR vaccination is associated with sub-clinical intestinal inflammation which is central to the autistic "enterocolitis" theory. The study was not designed to test directly the association of autism to MMR vaccination. MATERIAL AND METHODS: We studied 109/20 infants, before and two and four weeks after immunization with Pentavac and MMR vaccines, for the presence of intestinal inflammation (faecal calprotectin). RESULTS: Neither vaccination was associated with any significant increase in faecal calprotectin concentrations. CONCLUSIONS: The failure of the MMR vaccination to cause an intestinal inflammatory response provides evidence against the proposed gut-brain interaction that is central to the autistic "enterocolitis" hypothesis.

COX-1 and 2, intestinal integrity, and pathogenesis of nonsteroidal anti-inflammatory drug enteropathy in mice.

BACKGROUND & AIMS: The pathogenesis of nonsteroidal anti-inflammatory drug-induced enteropathy is controversial, but it is thought that cyclooxygenase-1 (COX-1) inhibition is of pivotal importance. We compared small intestinal function and morphology in untreated wild-type, COX-1- and COX-2-deficient mice and the effect of indomethacin, selective COX-1 (SC-560), and COX-2 (celecoxib) inhibition. METHODS: Intestinal permeability ((51)CrEDTA), inflammation (fecal granulocyte marker protein), prostaglandin E(2) (PGE(2)) levels, and macroscopic and microscopic appearances were assessed at baseline and after the drugs. RESULTS: COX-1(-/-) animals were normal except for a 97% decrease in intestinal PGE(2) levels. COX-1(+/+) and COX-1(-/-) animals reacted in a similar way to indomethacin. However, celecoxib, having caused no damage in COX-1(+/+) animals, caused small bowel ulcers in COX-1(-/-) animals. Selective inhibition of COX-1 decreased intestinal PGE(2) levels in COX-2(+/+) and COX-2(-/-) animals by 95%-97%, but caused only small bowel ulcers in the latter group. Dual inhibition of COX-1 and COX-2 in wild-type animals resulted in similar small bowel damage. Between 40% and 50% of untreated COX-2(-/-) animals had increased intestinal permeability and inflammation. Some had ileal ulcers that were distinctively different from indomethacin-induced ulcers. Furthermore, long-term celecoxib administration in wild-type animals was associated with similar damage as in the COX-2(-/-) mice. CONCLUSIONS: COX-1 deficiency or inhibition and short-term COX-2 inhibition are compatible with normal small intestinal integrity. Dual inhibition of the COX enzymes leads to damage similar to that seen with indomethacin. Long-term COX-2 deficiency or inhibition is associated with significant intestinal pathology despite normal intestinal PGE(2) levels, suggesting a role for COX-2 in the maintenance of small intestinal integrity in the mouse.

Subclinical intestinal inflammation and sacroiliac changes in relatives of patients with ankylosing spondylitis.

BACKGROUND & AIMS: It has been suggested that subclinical intestinal inflammation plays a pathogenic role in the spondylarthropathy of ankylosing spondylitis (AS). We assessed the possible presence and inheritance pattern of subclinical intestinal inflammation in first-degree relatives of patients with AS. The relationship between this inflammation and the subjects' HLA-B27 genotype as well as computerized tomographic sacroiliac abnormalities was also assessed. METHODS: A total of 124 of 213 (58%) available first-degree relatives of 47 patients with AS in Iceland underwent investigation for intestinal inflammation (fecal calprotectin concentration), HLA-B27 genotyping, and computerized tomography of the sacroiliac joints. RESULTS: A total of 41% of the first-degree relatives had subclinical intestinal inflammation, whereas 15 of 17 spouses were normal. Variance components analyses suggest that the inheritance pattern of this inflammation is affected by a major additive gene. Some sacroiliac changes, suggestive of early AS, differed significantly between subjects with and without subclinical intestinal inflammation (mean diameter of subchondral cysts [2.9 vs. 1.2 mm; P = 0.026] and blurring of joint margins [9 of 44 (20%) vs. 1 of 41 (2%); P = 0.02]). Intestinal inflammation and sacroiliac changes did not relate to the subjects' HLA-B27 status. CONCLUSIONS: Many first-degree relatives of patients with AS appear to have an inherited abnormality that leads to subclinical intestinal inflammation. The association between the presence of this inflammation and the sacroiliac changes suggests that it may play a pathogenic role in the spondylarthropathy of AS.

Subclinical intestinal inflammation: an inherited abnormality in Crohn's disease relatives?

BACKGROUND & AIMS: One approach to unraveling the genetics of complex inherited disease, such as Crohn's disease, is to search for subclinical disease markers among unaffected family members. We assessed the possible presence, prevalence, and inheritance pattern of subclinical intestinal inflammation in apparently healthy relatives of patients with Crohn's disease. METHODS: A total of 49 patients with Crohn's disease, 16 spouses, and 151 (58%) of 260 available first-degree relatives underwent a test for intestinal inflammation (fecal calprotectin concentration). The mode of inheritance was assessed from 36 index patients (by variance component analysis) when more than 50% of relatives were studied. RESULTS: Fecal calprotectin concentrations in patients with Crohn's disease (47 mg/L; confidence interval [CI], 27-95 mg/L) and relatives (11 mg/L; CI, 9-14 mg/L) differed significantly (P < 0.0001) from controls (4 mg/L; CI, 3-5 mg/L), whereas that of the spouses did not (4 mg/L; CI, 3-6 mg/L; P > 0.5). Fecal calprotectin concentration was increased in 49% of all relatives studied. The increased fecal calprotectin concentration among the relatives of the 36 index patients had an inheritance pattern that was most consistent with an additive inheritance pattern. CONCLUSIONS: There is a high prevalence of subclinical intestinal inflammation in first-degree relatives of patients with Crohn's disease that conforms best to an additive inheritance pattern. The genetic basis for this abnormality may represent a risk factor for Crohn's disease.