Assessing HDL Metabolism in Subjects with Elevated Levels of HDL Cholesterol and Coronary Artery Disease

High-density lipoprotein cholesterol (HDL-C) is thought to be atheroprotective yet some patients with elevated HDL-C levels develop cardiovascular disease, possibly due to the presence of dysfunctional HDL. We aimed to assess the metabolic fate of circulating HDL particles in patients with high HDL-C with and without coronary artery disease (CAD) using in vivo dual labeling of its cholesterol and protein moieties. We measured HDL apolipoprotein (apo) A-I, apoA-II, free cholesterol (FC), and cholesteryl ester (CE) kinetics using stable isotope-labeled tracers (D3-leucine and 13C2-acetate) as well as ex vivo cholesterol efflux to HDL in subjects with (n = 6) and without (n = 6) CAD that had HDL-C levels >90th percentile. Healthy controls with HDL-C within the normal range (n = 6) who underwent the same procedures were used as the reference. Subjects with high HDL-C with and without CAD had similar plasma lipid levels and similar apoA-I, apoA-II, HDL FC, and CE pool sizes with no significant differences in fractional clearance rates (FCRs) or production rates (PRs) of these components between groups. Subjects with high HDL-C with and without CAD also had similar basal and cAMP-stimulated ex vivo cholesterol efflux to HDL. When all subjects were considered (n = 18), unstimulated non-ABCA1-mediated efflux (but not ABCA1-specific efflux) was correlated positively with apoA-I production (r = 0.552, p = 0.017) and HDL FC and CE pool sizes, and negatively with the fractional clearance rate of FC (r = −0.759, p = 4.1 × 10−4) and CE (r = −0.652, p = 4.57 × 10−3). Our data are consistent with the concept that ex vivo non-ABCA1 efflux capacity may correlate with slower in vivo turnover of HDL cholesterol moieties. The use of a dual labeling protocol provided for the first time the opportunity to assess the association of ex vivo cholesterol efflux capacity with in vivo HDL cholesterol metabolic parameters.

Impaired Tubular Secretion of Organic Solutes in Advanced CKD

Background The clearance of solutes removed by tubular secretion may be altered out of proportion to the glomerular filtration rate (GFR) in chronic kidney disease (CKD). Recent studies have described considerable variability in the secretory clearance of waste solutes relative to the GFR in patients with CKD. Methods To test the hypothesis that secretory clearance relative to GFR is reduced in patients approaching dialysis, we used metabolomic analysis to identify solutes in simultaneous urine and plasma samples from 16 CKD patients with an estimated GFR of 7±2 ml/min per 1.73m2 and 16 control participants. Fractional clearances were calculated as the ratios of urine to plasma levels of each solute relative to those of creatinine and urea in CKD patients and to those of creatinine in controls. Results Metabolomic analysis identified 39 secreted solutes with fractional clearance >3.0 in control participants. Fractional clearance values in CKD patients were reduced on average to 65%±27% of those in controls. These values were significantly lower for 18 of 39 individual solutes and significantly higher for only one. Assays of the secreted anions phenylacetyl glutamine, p-cresol sulfate, indoxyl sulfate, and hippurate confirmed variable impairment of secretory clearances in advanced CKD. Fractional clearances were markedly reduced for phenylacetylglutamine (4.2±0.6 for controls versus 2.3±0.6 for CKD patients, P<0.001), p-cresol sulfate (8.6±2.6 for controls versus 4.1±1.5 for CKD patients, P<0.001), and indoxyl sulfate (23.0±7.3 versus 7.5±2.8, P<0.001), but not for hippurate (10.2±3.8 versus 8.4±2.6, P=0.13). Conclusions Secretory clearances for many solutes are reduced more relative to the reduction in GFR in advanced CKD. Impaired secretion of these solutes might contribute to uremic symptoms as patients approach dialysis.

Impaired Tubular Secretion of Organic Solutes in Acute Kidney Injury

BackgroundImpairment of kidney function is routinely assessed by measuring the accumulation of creatinine, an organic solute cleared largely by glomerular filtration. We tested whether the clearance of solutes that undergo tubular secretion is reduced in proportion to the clearance of creatinine in humans with AKI.MethodsFour endogenously produced organic solutes (phenylacetylglutamine [PAG], hippurate [HIPP], indoxyl sulfate [IS], and p-cresol sulfate [PCS]) were measured in spot urine and plasma samples from ten patients with AKI and 17 controls. Fractional clearance relative to creatinine was calculated to assess tubular secretion. Fractional clearance values were calculated in terms of the free, unbound levels of HIPP, IS, and PCS that bind to plasma proteins.ResultsFractional clearance values for PAG, HIPP, IS, and PCS were >1.0 in patients with AKI as well as controls, indicating that these solutes were still secreted by the tubules of the injured kidneys. Fractional clearance values were, however, significantly lower in patients with AKI than controls, indicating that kidney injury reduced tubular secretion more than glomerular filtration (AKI versus control: PAG, 2.1±0.7 versus 4.6±1.4, P<0.001; HIPP, 10±5 versus 15±7, P=0.02; IS, 10±6 versus 28±7, P<0.001; PCS, 3.3±1.8 versus 10±3, P<0.001). Free plasma levels rose out of proportion to total plasma levels for each of the bound solutes in AKI, so that calculating their fractional clearance in terms of their total plasma levels failed to reveal their impaired secretion.ConclusionsTubular secretion of organic solutes can be reduced out of proportion to glomerular filtration in AKI. Impaired secretion of protein-bound solutes may be more reliably detected when clearances are expressed in terms of their free, unbound levels in the plasma.

P0105THE ROLE OF PROTEOGLYCANS IN GLOMERULAR PHYSIOLOGY AND PATHOPHYSIOLOGY

Background and Aims Damage to the filtration barrier in the kidney may result in proteinuria, a hallmark of kidney disease. The glomerular filtration barrier consist of 3 layers: the endothelial cells with their endothelial surface layer (ESL), the glomerular basement membrane and podocytes. The ESL is rich in negatively charged molecules, such as proteoglycans (PGs). The negatively charged molecules restrain the flow of charged molecules, as albumin, over the barrier. Loss of ESL has been shown to lead to proteinuria without damage to the other structures of the filtration barrier. In order to gain further knowledge about the function and composition of the glomerular ESL we eluted the glomerular ESL from rats and analyzed the PG content as well as the effect of loss of the ESL on the function of the glomerular filtration. Method The basic principles of ion exchange chromatography were applied in this study. In order to elute highly negatively charged proteoglycans of the ESL, we used 1 M NaCl solution (HS). 1 M mannitol (HO) was included to evaluate osmotic effects. A control fraction was eluted with normal saline solution (0,15 M NaCl) (NS). Solutions were introduced intraarterially to rat kidneys under anesthesia in vivo. Venous effluent was collected and analyzed using proteomics and mass spectrometry. Fractional clearance of albumin was measured as well as GFR. Electron microscopy (EM) was used to investigate changes to the filtration barrier and the thickness of the glomerular ESL. Immunohistochemistry was used to confirm the presence of identified PGs in human tissue. Results We identified 15 PGs and PG candidates in ESL as well as hyaluronan. The most abundant PGs were lumican, glypican-1, syndecan-4, perlecan, podocan, decorin, serglycan, agrin and biglycan. In general, PGs were found in in all HS, NS and HO fractions, but in higher yields in the HS samples. EM demonstrated that the glomerular ESL thickness was significantly reduced in HS perfused rats – 28% (p&lt;0.05) compared to rats perfused with NS. Rats perfused with HS also had increased fractional clearance of albumin compared to NS and HO, and a large reduction of GFR 10 minutes after perfusion. Conclusion The ESL itself represents a dynamic structure with significant molecular turnover and is formed by PGs, glycosaminoglycans, glycoproteins and soluble proteins. In our study, we identified several PGs in the glomerular endothelial ESL. We show that loss of ESL leads to increased albumin fractional clearance and reduction in GFR after perfusion, strongly suggesting that the ESL is important for preventing proteinuria. Characterizing the composition of the glomerular ESL is therefore of great importance, and can give new possible targets for treatment of proteinuria.

Urinary Fractional Clearance of Sodium in 8 Healthy Beagle Dogs Fed Normal, Low, or Ultralow Sodium Diets

The purpose of this study was to investigate in healthy adult dogs if there was a daily fluctuation in the FCNa, the role that dietary sodium intake played on the FCNa, and the role that feeding played on the obtained value for FCNa. Three different diets were used in a group of 8 healthy beagle dogs in a crossover design. The sodium content of the diets was normal (0.26%), low (0.18%), and ultralow (0.06%). Spot urine and blood samples were collected from which the urine and serum sodium and creatinine concentration were determined, and the FCNa was calculated. The median FCNa for the normal, low, and ultralow sodium diets was 0.5, 0.77, and 0.15, respectively. Individual dogs showed a daily variation in FCNa, and samples which were collected shortly after eating showed the greatest variation. This study showed that in a group of healthy beagle dogs without obvious renal disease, the FCNa could exceed 1 and that there was both an individual and daily variation in the FCNa. The greatest variation was seen whilst the dogs were fed the low and ultralow sodium diets and when the samples were collected shortly after eating. This study concluded that an FCNa > 1% may not be indicative of acute tubular dysfunction in young dogs, and use of the FCNa for assessing renal function in clinical cases should take into account the animal’s diet, as well as the time the samples were taken in relation to feeding.

Is N,N-dimethylglycine N-oxide a choline and betaine metabolite?

Choline metabolism is by oxidation to betaine, which is demethylated to N,N-dimethylglycine; dimethylglycine is oxidatively demethylated to sarcosine. This pathway is important for osmoregulation and as a source of methyl groups. We asked whether another metabolite was involved. We synthesized the N-oxide of dimethylglycine (DMGO) by oxidizing dimethylglycine with peracetic acid, and measured DMGO in human plasma and urine by HPLC-MS/MS with positive ion detection, using two chromatography procedures, based on ion exchange and HILIC separations. The molecular ion DMGOH+ (m/z=120) yielded four significant fragments (m/z=103, 102, 58 and 42). The suspected DMGO peak in human body fluids showed all these fragments, and co-chromatographed with added standard DMGO in both HPLC systems. Typical plasma concentrations of DMGO are under 1 μmol/l. They may be lower in metabolic syndrome patients. Urine concentrations are higher, and DMGO has a higher fractional clearance than dimethylglycine, betaine and choline. It was present in all of over 80 human urine and plasma samples assayed. Plasma DMGO concentrations correlate with plasma DMG concentrations, with betaine and choline concentrations, with the osmolyte myo-inositol, and strongly with urinary DMGO excretion. We conclude that DMGO is probably a normal human metabolite.

Abstract 18390: Effects of a Proprotein Convertase Subtilisin/kexin Type 9 (PCSK9) Inhibitor, Alirocumab, on Lipid and Lipoprotein Metabolism in Healthy Subjects

Background: Alirocumab is a monoclonal antibody that inhibits binding of PCSK9 to LDL receptors, decreasing their lysosomal degradation and increasing their numbers on cell surfaces. In Phase 2/3 studies, alirocumab 150 mg lowered LDL-cholesterol (C) ~61%, apoB 53%, and fasting triglycerides (TG) 16%. The mechanisms that increased LDL removal or the effects on postprandial (PP) TG levels after alirocumab therapy have not been reported. Method: 18 (10F, 8 M), healthy volunteers completed a Phase 1, placebo-controlled, single-blind, single-sequence study. Subjects received 2 placebo doses followed by 5 alirocumab doses, 150 mg SC every 2 wks. At the end of each treatment period, we measured fasting lipids and lipoproteins and subjects received a high fat meal with PP TG and apoB48 levels measured over 8hrs. Stable isotope studies were performed 3-5 days later to measure apoB turnover in very low density (VLDL), intermediate density (IDL) and LDL. Results: In 18 subjects, Alirocumab significantly reduced plasma total-C by 35% (172±32 to 111±24 mg/dL), LDL-C by 60% (104±23 to 44±21 mg/dL) and, apoB by 45% (90±21 to 45±12 mg/dL). Plasma TG levels decreased by 13% (99±44 to 86±40mg/dL). LDL-C, LDL-TG, and LDL-apoB isolated from plasma by ultracentrifugation fell by 50±15%, 5±2%, and 41±16%, respectively, on alirocumab. Preliminary Kinetic results on N=10, show that the reductions in LDL-apoB were due to an increase in the fractional clearance rate (FCR) of LDL-apoB from 0.50±0.18 on placebo to 1.02±0.35 pools/day on alirocumab (p<0.001). IDL-C, IDL-TG and IDL-apoB were also reduced significantly, with a trend toward an increase in IDL-apoB FCR on alirocumab (placebo: 9.2±4 vs alirocumab: 10.8±3 pools/day; p=0.06). Alirocumab had no effects VLDL-C, VLDL-TG, or VLDL-apoB, nor did alirocumab change VLDL FCR or PR. The area under the curve for PP TG and PP apoB48 did not change from placebo to alirocumab. No serious adverse event or treatment discontinuation occurred. Results for all 18 subjects will be available for presentation. Summary: Alirocumab treatment significantly reduced levels of IDL and LDL-apoB by increasing the FCRs of these lipoproteins, particularly LDL. Alirocumab did not alter the metabolism of VLDL-apoB or PP TG-rich lipoproteins.