Differences in faecal microbiome composition between adult patients with UCD and PKU and healthy control subjects.

Urea cycle disorders (UCDs) are a group of rare inherited metabolic diseases causing hyperammonemic encephalopathy. Despite intensive dietary and pharmacological therapy, outcome is poor in a subset of UCD patients. Reducing ammonia production by changing faecal microbiome in UCD is an attractive treatment approach. We compared faecal microbiome composition of 10 UCD patients, 10 healthy control subjects and 10 phenylketonuria (PKU) patients. PKU patients on a low protein diet were included to differentiate between the effect of a low protein diet and the UCD itself on microbial composition. Participants were asked to collect a faecal sample and to fill out a 24 h dietary journal. DNA was extracted from faecal material, taxonomy was assigned and microbiome data was analyzed, with a focus on microbiota involved in ammonia metabolism.In this study we show an altered faecal microbiome in UCD patients, different from both PKU and healthy controls. UCD patients on dietary and pharmacological treatment had a less diverse faecal microbiome, and the faecal microbiome of PKU patients on a protein restricted diet with amino acid supplementation showed reduced richness compared to healthy adults without a specific diet. The differences in the microbiome composition of UCD patients compared to healthy controls were in part related to lactulose use. Other genomic process encodings involved in ammonia metabolism, did not seem to differ. Since manipulation of the microbiome is possible, this could be a potential treatment modality. We propose as a first next step, to study the impact of these faecal microbiome alterations on metabolic stability. TAKE HOME MESSAGE: The faecal microbiome of UCD patients was less diverse compared to PKU patients and even more compared to healthy controls.

Proteinuria in early childhood due to familial LCAT deficiency caused by loss of a disulfide bond in lecithin:cholesterol acyl transferase.

INTRODUCTION: Familial lecithin:cholesterol acyltransferase (LCAT) deficiency (FLD) is a rare recessive disorder of cholesterol metabolism characterized by the absence of high density lipoprotein (HDL) and the triad of corneal opacification, hemolytic anemia and glomerulopathy. PATIENTS: We here report on FLD in three siblings of a kindred of Moroccan descent with HDL deficiency. In all cases (17, 12 and 3 years of age) corneal opacification and proteinuria were observed. In the 17-year-old female proband, anemia with target cells was observed. RESULTS: Homozygosity for a mutation in LCAT resulted in the exchange of cysteine to tyrosine at position 337, disrupting the second disulfide bond in LCAT. LCAT protein and activity were undetectable in the patients' plasma and in media of COS7 cells transfected with an expression vector with mutant LCAT cDNA. Upon treatment with an ACE inhibitor and a thiazide diuretic, proteinuria in the proband decreased from 6g to 2g/24h. CONCLUSION: This is the first report that FLD can cause nephropathy at a very early age.

Improvement of lipid profile is accompanied by atheroprotective alterations in high-density lipoprotein composition upon tumor necrosis factor blockade: a prospective cohort study in ankylosing spondylitis.

OBJECTIVE: Cardiovascular mortality is increased in ankylosing spondylitis (AS), and inflammation plays an important role. Inflammation deteriorates the lipid profile and alters high-density lipoprotein cholesterol (HDL-c) composition, reflected by increased concentrations of serum amyloid A (SAA) within the particle. Anti-tumor necrosis factor (anti-TNF) treatment may improve these parameters. We therefore undertook the present study to investigate the effects of etanercept on lipid profile and HDL composition in AS. METHODS: In 92 AS patients, lipid levels and their association with the inflammation markers C-reactive protein (CRP), erythrocyte sedimentation rate, and SAA were evaluated serially during 3 months of etanercept treatment. HDL composition and its relationship to inflammation markers was determined in a subgroup of patients, using surface-enhanced laser desorption/ionization time-of-flight analysis. RESULTS: With anti-TNF treatment, levels of all parameters of inflammation decreased significantly, whereas total cholesterol, HDL-c, and apolipoprotein A-I (Apo A-I) levels increased significantly. This resulted in a better total cholesterol:HDL-c ratio (from 3.9 to 3.7) (although the difference was not statistically significant), and an improved Apo B:Apo A-I ratio, which decreased by 7.5% over time (P=0.008). In general, increases in levels of all lipid parameters were associated with reductions in inflammatory activity. In addition, SAA was present at high levels within HDL particles from AS patients with increased CRP levels and disappeared during treatment, in parallel with declining plasma levels of SAA. CONCLUSION: Our results show for the first time that during anti-TNF therapy for AS, along with favorable changes in the lipid profile, HDL composition is actually altered whereby SAA disappears from the HDL particle, increasing its atheroprotective ability. These findings demonstrate the importance of understanding the role of functional characteristics of HDL-c in cardiovascular diseases related to chronic inflammatory conditions.

ApoAI-phosphatidylcholine infusion neutralizes the atherothrombotic effects of C-reactive protein in humans.

BACKGROUND: High-density lipoprotein (HDL) exerts a variety of anti-atherothrombotic functions, including a potent anti-inflammatory impact. In line, the direct pro-inflammatory effects of C-reactive protein (CRP) can be attenuated by HDL in vitro. OBJECTIVE: To evaluate whether this also holds true in humans, we assessed the ability of reconstituted HDL to neutralize CRP-mediated activation of coagulation and inflammation. METHODS: Fifteen healthy male volunteers received an infusion of recombinant human (rh)CRP (1.25 mg kg(-1) body weight). In eight of these volunteers, an infusion of human apoAI reconstituted with phosphatidylcholine (apoAI-PC; 80 mg kg(-1) body weight) preceded rhCRP infusion. RESULTS: Infusion of rhCRP alone elicited an inflammatory response and thrombin generation. In individuals who received apoAI-PC prior to rhCRP, these effects were abolished. Parallel tests in primary human endothelial cells showed that apoAI-PC preincubation with rhCRP abolished the CRP-mediated activation of inflammation as assessed by IL-6 release. Although we were able to show that rhCRP co-eluted with HDL after size-exclusion chromatography, plasmon surface resonance indicated the absence of a direct interaction between HDL and CRP. CONCLUSION: Infusion of apoAI-PC prior to rhCRP in humans completely prevents the direct atherothrombotic effects of rhCRP. These findings imply that administration of apoAI-PC may offer benefit in patients with increased CRP.

Perioperative hyperinsulinaemic normoglycaemic clamp causes hypolipidaemia after coronary artery surgery.

BACKGROUND: Glucose-insulin-potassium (GIK) administration is advocated on the premise of preventing hyperglycaemia and hyperlipidaemia during reperfusion after cardiac interventions. Current research has focused on hyperglycaemia, largely ignoring lipids, or other substrates. The present study examines lipids and other substrates during and after on-pump coronary artery bypass grafting and how they are affected by a hyperinsulinaemic normoglycaemic clamp. METHODS: Forty-four patients were randomized to a control group (n=21) or to a GIK group (n=23) receiving a hyperinsulinaemic normoglycaemic clamp during 26 h. Plasma levels of free fatty acid (FFA), total and lipoprotein (VLDL, HDL, and LDL)-triglycerides (TG), ketone bodies, and lactate were determined. RESULTS: In the control group, mean FFA peaked at 0.76 (sem 0.05) mmol litre(-1) at early reperfusion and decreased to 0.3-0.5 mmol litre(-1) during the remaining part of the study. GIK decreased FFA levels to 0.38 (0.05) mmol litre(-1) at early reperfusion, and to low concentrations of 0.10 (0.01) mmol litre(-1) during the hyperinsulinaemic clamp. GIK reduced the area under the curve (AUC) for FFA by 75% and for TG by 53%. The reduction in total TG was reflected by a reduction in the VLDL (-54% AUC) and HDL (-42% AUC) fraction, but not in the LDL fraction. GIK prevented the increase in ketone bodies after reperfusion (-44 to -47% AUC), but was without effect on lactate levels. CONCLUSIONS: Mild hyperlipidaemia was only observed during early reperfusion (before heparin reversal) and the hyperinsulinaemic normoglycaemic clamp actually resulted in hypolipidaemia during the largest part of reperfusion after cardiac surgery.

Lipopolysaccharide is transferred from high-density to low-density lipoproteins by lipopolysaccharide-binding protein and phospholipid transfer protein.

Lipopolysaccharide (LPS), the major outer membrane component of gram-negative bacteria, is a potent endotoxin that triggers cytokine-mediated systemic inflammatory responses in the host. Plasma lipoproteins are capable of LPS sequestration, thereby attenuating the host response to infection, but ensuing dyslipidemia severely compromises this host defense mechanism. We have recently reported that Escherichia coli J5 and Re595 LPS chemotypes that contain relatively short O-antigen polysaccharide side chains are efficiently redistributed from high-density lipoproteins (HDL) to other lipoprotein subclasses in normal human whole blood (ex vivo). In this study, we examined the role of the acute-phase proteins LPS-binding protein (LBP) and phospholipid transfer protein (PLTP) in this process. By the use of isolated HDL containing fluorescent J5 LPS, the redistribution of endotoxin among the major lipoprotein subclasses in a model system was determined by gel permeation chromatography. The kinetics of LPS and lipid particle interactions were determined by using Biacore analysis. LBP and PLTP were found to transfer LPS from HDL predominantly to low-density lipoproteins (LDL), in a time- and dose-dependent manner, to induce remodeling of HDL into two subpopulations as a consequence of the LPS transfer and to enhance the steady-state association of LDL with HDL in a dose-dependent fashion. The presence of LPS on HDL further enhanced LBP-dependent interactions of LDL with HDL and increased the stability of the HDL-LDL complexes. We postulate that HDL remodeling induced by LBP- and PLTP-mediated LPS transfer may contribute to the plasma lipoprotein dyslipidemia characteristic of the acute-phase response to infection.

Apolipoprotein A-V: a novel apolipoprotein associated with an early phase of liver regeneration.

Liver regeneration in response to various forms of liver injury is a complex process, which ultimately results in restoration of the original liver mass and function. Because the underlying mechanisms that initiate this response are still incompletely defined, this study was aimed to identify novel factors. Liver genes that were up-regulated 6 h after 70% hepatectomy (PHx) in the rat were selected by cDNA subtractive hybridization. Besides known genes associated with cell proliferation, several novel genes were isolated. The novel gene that was most up-regulated was further studied. Its mRNA showed a liver-specific expression and encoded a protein comprising 367 amino acids. The mouse and human cDNA analogues were also isolated and appeared to be highly homologous. The human gene analogue was located at an apolipoprotein gene cluster on chromosome 11q23. The protein encoded by this gene had appreciable homology with apolipoproteins A-I and A-IV. Maximal expression of the gene in the rat liver and its gene product in rat plasma was observed 6 h after PHx. The protein was present in plasma fractions containing high density lipoprotein particles. Therefore, we have identified a novel apolipoprotein, designated apolipoprotein A-V, that is associated with an early phase of liver regeneration.

Distribution and kinetics of lipoprotein-bound endotoxin.

Lipopolysaccharide (LPS), the major glycolipid component of gram-negative bacterial outer membranes, is a potent endotoxin responsible for pathophysiological symptoms characteristic of infection. The observation that the majority of LPS is found in association with plasma lipoproteins has prompted the suggestion that sequestering of LPS by lipid particles may form an integral part of a humoral detoxification mechanism. Previous studies on the biological properties of isolated lipoproteins used differential ultracentrifugation to separate the major subclasses. To preserve the integrity of the lipoproteins, we have analyzed the LPS distribution, specificity, binding capacity, and kinetics of binding to lipoproteins in human whole blood or plasma by using high-performance gel permeation chromatography and fluorescent LPS of three different chemotypes. The average distribution of O111:B4, J5, or Re595 LPS in whole blood from 10 human volunteers was 60% (+/-8%) high-density lipoprotein (HDL), 25% (+/-7%) low-density lipoprotein, and 12% (+/-5%) very low density lipoprotein. The saturation capacity of lipoproteins for all three LPS chemotypes was in excess of 200 microg/ml. Kinetic analysis however, revealed a strict chemotype dependence. The binding of Re595 or J5 LPS was essentially complete within 10 min, and subsequent redistribution among the lipoprotein subclasses occurred to attain similar distributions as O111:B4 LPS at 40 min. We conclude that under simulated physiological conditions, the binding of LPS to lipoproteins is highly specific, HDL has the highest binding capacity for LPS, the saturation capacity of lipoproteins for endotoxin far exceeds the LPS concentrations measured in clinical situations, and the kinetics of LPS association with lipoproteins display chemotype-dependent differences.

Detection and clinical relevance of human endotoxemia.

An optimized chromogenic assay for the detection of endotoxins in human blood is described. The assay comprises the removal of the inhibitory activity of plasma components by a dilution plus heating procedure, endotoxin-dependent activation of Limulus amebocyte lysate, and chromogenic measurement of the activated lysate. The assay has a detection limit of 3 ng endotoxin/L plasma. At the 10 ng/L level within-assay CV and between-assay CV of 14 and 21% were obtained respectively. In a prospective clinical trial including 473 consecutive febrile patients the assay has previously been demonstrated to have positive and negative predictive values of 48% and 99% for impending Gram-negative sepsis, respectively. In a similar study in 76 consecutive patients with Gram-negative infection of the urinary tract, these values were 73% and 95%, respectively. We conclude that this assay may provide the means to select those patients who are most likely to benefit from anti-endotoxin treatment. To facilitate endotoxin testing in other laboratories, a preliminary evaluation of a commercial endotoxin assay versus our own method was performed with 108 duplicate blood samples obtained from septic patients. With this assay detection limits of 2-3 ng endotoxin/L plasma could be obtained, as well as a good correlation (r = 0.94) and level of consensus to establish endotoxemia (93%) as compared to the house method. The commercial assay may therefore facilitate the introduction of endotoxin testing in other laboratories.