Fractional Clearance Research Articles

The Pharmacokinetics, Metabolism, and Clearance Mechanisms of Ritlecitinib, a Janus Kinase 3 and Tyrosine-Protein Kinase Family Inhibitor, in Humans.

Ritlecitinib is an oral once-daily irreversible inhibitor of Janus kinase 3 and tyrosine-protein kinase family being developed for the treatment of moderate-to-severe alopecia areata. This study examined the disposition of ritlecitinib in male participants following oral and intravenous administration using accelerator mass spectroscopy methodology to estimate pharmacokinetic parameters and characterize metabolite profiles. The results indicated ritlecitinib had a systemic clearance of 43.7 L/h, a steady state volume of distribution of 73.8 L, extent of absorption of 89%, time to maximum plasma concentration of ∼0.5 hours, and absolute oral bioavailability of 64%. An observed long terminal half-life of total radioactivity was primarily attributed to ritlecitinib binding to plasma albumin. Ritlecitinib was the main circulating drug species in plasma (∼30%), with one major pharmacologically inactive cysteine conjugated metabolite (M2) at >10%. Oxidative metabolism (fractional clearance 0.47) and glutathione-related conjugation (fractional clearance 0.24) were the primary routes of elimination for ritlecitinib with the greatest disposition of radioactivity shown in the urine (∼71%). In vitro phenotyping indicated ritlecitinib cytochrome P450 (CYP) fraction of metabolism assignments of 0.29 for CYP3A, 0.09 for CYP2C8, 0.07 for CYP1A2, and 0.02 for CYP2C9. In vitro phenotyping in recombinant human glutathione S-transferases indicated ritlecitinib was turned over by a number of cytosolic and microsomal enzyme isoforms. SIGNIFICANCE STATEMENT: This study provides a detailed understanding of the disposition and metabolism of ritlecitinib, a JAK3 and TEC family kinase inhibitor for alopecia areata in humans, as well as characterization of clearance pathways and pharmacokinetics of ritlecitinib and its metabolites. As an AMS-based ADME study design, we have expanded on reporting the standard ADME endpoints, providing key pharmacokinetic parameters, such as clearance, volume of distribution, and bioavailability, allowing for a more comprehensive understanding of drug disposition.

Modelling and assessment of glucose-lactate kinetics in youth withoverweight, obesityand metabolic dysfunction-associated steatotic liver disease: A pilot study.

In this study, we investigated glucose and lactate kinetics during a 75 g oral glucose tolerance test (OGTT) in 23 overweight and obese adolescents and assessed putative differences among participants with and without metabolic dysfunction-associated steatotic liver disease (MASLD). We enrolled 23 young people (six girls) with obesity [body mass index 33 (29-37)]. Glucose-lactate kinetics parameters (disposal glucose insulin sensitivity, SID; fraction of glucose converted into lactate, fr; fractional lactate clearance rate, kL) and lactate production rate (LPR) were estimated using the oral glucose-lactate minimal model. MASLD presence was assessed using the proton density fat fraction. We analysed glucose, lactate and LPR time to peak, peak values and area under the curve and evaluated differences using the Wilcoxon test. MASLD and no-MASLD participants were compared using the Mann-Whitney test. Correlations between parameters were assessed using the Spearman correlation coefficient (ρ). We also tested the performance of two (4 or 3 h OGTT) protocols in estimating oral glucose-lactate minimal model and LPR parameters. Glucose peaks 30 min earlier than lactate (p = .0019). This pattern was present in the no-MASLD group (p < .001). LPR peaks 30 min later in the MASLD group (p = .02). LPR and kL were higher in MASLD, suggesting higher glycolysis and lactate utilization. SID and fr correlate significantly (ρ = -0.55, p = .008). SID and fr were also correlated with the body mass index, (ρ = -0.45, p = .04; and ρ = 0.45; p = .03). The protocol duration did not influence the estimates of the parameters. Youth with MASLD showed a delayed glucose metabolism, possibly because of greater utilization of the underlying substrates. A 3-h OGTT may be used to assess lactate metabolism effectively.

Effects of Recombinant Human Lecithin Cholesterol Acyltransferase on Lipoprotein Metabolism in Humans.

LCAT (lecithin cholesterol acyl transferase) catalyzes the conversion of unesterified, or free cholesterol, to cholesteryl ester, which moves from the surface of HDL (high-density lipoprotein) into the neutral lipid core. As this iterative process continues, nascent lipid-poor HDL is converted to a series of larger, spherical cholesteryl ester-enriched HDL particles that can be cleared by the liver in a process that has been termed reverse cholesterol transport. We conducted a randomized, placebocontrolled, crossover study in 5 volunteers with atherosclerotic cardiovascular disease, to examine the effects of an acute increase of recombinant human (rh) LCAT via intravenous administration (300-mg loading dose followed by 150 mg at 48 hours) on the in vivo metabolism of HDL APO (apolipoprotein)A1 and APOA2, and the APOB100-lipoproteins, very low density, intermediate density, and low-density lipoproteins. As expected, recombinant human LCAT treatment significantly increased HDL-cholesterol (34.9 mg/dL; P≤0.001), and this was mostly due to the increase in cholesteryl ester content (33.0 mg/dL; P=0.014). This change did not affect the fractional clearance or production rates of HDL-APOA1 and HDL-APOA2. There were also no significant changes in the metabolism of APOB100-lipoproteins. Our results suggest that an acute increase in LCAT activity drives greater flux of cholesteryl ester through the reverse cholesterol transport pathway without significantly altering the clearance and production of the main HDL proteins and without affecting the metabolism of APOB100-lipoproteins. Long-term elevations of LCAT might, therefore, have beneficial effects on total body cholesterol balance and atherogenesis.

A targeted proteomics method for quantifying plasma apolipoprotein kinetics in individual mice using stable isotope labeling

Altered apolipoprotein kinetics play a critical role in promoting dyslipidemia and atherogenesis. Human apolipoprotein kinetics have been extensively evaluated, but similar studies in mice are hampered by the lack of robust methods suitable for the small amounts of blood that can be collected at sequential time points from individual mice. We describe a targeted liquid chromatography tandem mass spectrometry method for simultaneously quantifying the stable isotope enrichment of several apolipoproteins represented by multiple peptides in serial blood samples (15 μl each) obtained after retro-orbital injection of 13C6,15N2-lysine (Lys8) in mice. We determined apolipoprotein fractional clearance rates (FCRs) and production rates (PRs) in WT mice and in two genetic models widely used for atherosclerosis research, LDL receptor–deficient (Ldlr−/−) and apolipoprotein E–deficient (Apoe−/−) mice. Injection of Lys8 produced a unique and readily detectable mass shift of labeled compared with unlabeled peptides with sensitivity allowing robust kinetics analyses. Ldlr−/− mice showed slower FCRs of APOA1, APOA4, total APOB, APOB100, APOCs, APOE and APOM, while FCRs of APOA1, APOB100, APOC2, APOC3, and APOM were not lower in Apoe−/− mice versus WT mice. APOE PR was increased in Ldlr−/− mice, and APOB100 and APOA4 PRs were reduced in Apoe−/− mice. Thus, our method reproducibly quantifies plasma apolipoprotein kinetics in different mouse models. The method can easily be expanded to include a wide range of proteins in the same biospecimen and should be useful for determining the kinetics of apolipoproteins in animal models of human disease.

Solução intravenosa poliiônica com concentração elevada de lactato (84 mEq/L) é eficaz para o tratamento de bezerros com diarreia

ABSTRACT This study aimed to evaluate the effectiveness of an electrolyte solution with a high lactate concentration (84mEq/L; L84) in correcting imbalances in diarrheal calves. Another solution with similar electrolyte composition, containing 84mEq/L of bicarbonate (B84) was used for comparison. Diarrhea was induced in neonatal calves by rotavirus infection, sucrose ingestion and furosemide administration. When a moderate degree of metabolic acidosis was reached, the calves were treated with intravenous infusion of L84 (n=10) or B84 (n=10) solutions. Venous blood and urine samples were collected before starting the infusion (0 h), in the middle (2.5 h), at the end (5.0 h) and 2.5 hours later. Total plasma protein concentration, blood and urine pH, blood pCO2, HCO3 -, and BE, plasma and urine concentration of L-lactate, and serum and urine concentrations of Na+, K+, and Cl- were measured. Strong ion difference, total concentration of non-volatile weak acids, anion gap, strong ion gap, percentage change in plasma volume, and urinary fractional clearances of Na+, K+, Cl-, and L-lactate were calculated. The calves demonstrated moderate dehydration, hypernatremia, hyperchloremia, metabolic acidosis, and unmeasured anion accumulation. These changes were completely corrected with the L84 solution, and the effects did not differ from those observed with B84 solution. The calves recovered without further therapeutic procedures, demonstrating that the L84 solution was effective for treating diarrheal calves.

Analysis of parameters of purine metabolism in patients with diabetes mellitus type 2

The aim of this research is to comprehensively analyze the current state of purine metabolism as well as assess its features in patients with diabetes mellitus type 2, taking into account the clinical and metabolic polymorphism of the disease. The authors examined 327 patients with diabetes mellitus type 2 (144 men and 183 women), a group of individuals with an average age of 57.8±10.1 years. The average duration of the disease was (9.3±7.3) years. All subjects received oral hypoglycemic therapy. The researchers determined anthropometric indicators, indicators of carbohydrate metabolism, concentration of creatinine, uric acid, parameters of the blood lipid spectrum, purine bases, activity of xanthine oxidase and hypoxanthine-guanine-phosphoribosyltransferase in the blood. In the course of the study the authors verified excretion of uric acid and creatinine renal uric acid, fractional clearance of uric acid, total tubular reabsorption of uric acid and coefficient of complete degradation of purine bases. Using the software complex "Statgraphics Centurion 18.0", we statistically analyzed the obtained data. The research showed that a decrease in assimilation, increased oxidation of purine bases due to the activation of xanthine oxidase, and a decrease in their reutilization by inhibiting the activity of hypoxanthine-guanine-phospho­ribo­syltransferase cause the high intensity of purine catabolism in patients with diabetes mellitus type 2. Hyperuricemia (28.0%), increased creatinine renal uric acid (62.3%), increased coefficient of complete degradation of purine bases (41.6%), activity of xanthine oxidase (38.8%) and inhibition of hypoxanthine-guanine-phosphoribosyltransferase activity (50.3%) dominate in the structure of disorders of purine bases in patients with diabetes mellitus type 2. We found that more than 50% of the subjects had high concentrations of purine bases in the blood. Purine bases concentrations in the blood of patients with diabetes mellitus type 2 closely correlate with each other and with the activity of xanthine oxidase and do not correlate with the level of uricemia, the level of uricemia does not correlate with the level of uric acid excretion. That is, the concentration of uric acid in the blood of patients with diabetes mellitus type 2 does not objectively reflect its production in the body. Patients with diabetes mellitus type 2 with obesity associate hyperuricemia with significantly lower creatinine renal uric acid and fractional clearance of uric acid as well as activation of the anabolic pathway of purine bases deposition.

Choroid plexus protein turnover in human choroid plexus organoids recapitulates turnover in humans measured using stable isotope labeling kinetics (SILK)

AbstractBackgroundThe choroid plexus secretes protein‐rich cerebrospinal fluid (CSF). CSF turnover is a key mechanism for protein clearance. Neurodegenerative diseases such as AD are characterised by accumulation of excess proteins that form pathological inclusions. CSF flow dysregulation could therefore be involved in the pathophysiology of neurodegenerative diseases (ND) such as Alzheimer’s Disease. A method for measuring CSF turnover in vivo may be achieved by quantitating CSF derived protein kinetics, providing a biomarker tool with to interrogate the relationship between CSF flow disequilibrium and neurodegeneration.MethodUsing untargeted proteomics we identify the most abundant proteins in a) stem‐cell derived choroid plexus organoids and b) human CSF. We then develop targeted mass spectrometry methods to quantitate protein specific peptides in both matrices. Using a stable isotope (13C6 leucine) we label a) choroid plexus organoids and b) humans with suspected NPH undergoing a 72 hour lumbar drainage protocol. We collect serial samples of the CSF‐like organoid fluid and CSF. We then measure the ratio of labelled to unlabelled peptides to quantitate the rates of protein labelling (fractional synthesis rate) and label loss (fractional clearance rate), defining the protein turnover rate.ResultHighly‐expressed proteins in human CSF from nine individuals were also present in CSF‐like fluid from CP organoids. Of these common proteins, we were able to quantitate the turnover rates of transthyretin, apolipoprotein E, cystatin C and pigment epithelium‐derived factor, and the synthesis rates of serotransferrin and albumin. We will correlate these findings with clinical measures of CSF production.ConclusionChoroid plexus protein synthesis and clearance can be measured using SILK; choroid plexus organoid fluid protein turnover recapitulates that seen in human CSF.

Loss of Glomerular Permselectivity in Type 2 Diabetes Associates With Progression to Kidney Failure.

We examined whether defects in glomerular size selectivity in type 2 diabetes (T2D) associated with progressive kidney disease. Glomerular filtration rate (GFR) and fractional clearances of dextrans of graded sizes were measured in 185 Pima Indians. The permselectivity model that best fit the dextran sieving data represented the glomerular capillary as being perforated by small restrictive pores and a parallel population of larger non-restrictive pores characterized by ωo, the fraction of total filtrate volume passing through this shunt. Hazard ratio (HR) for kidney failure was expressed per 1 standard deviation (SD) increase of ωo by Cox regression after adjusting for age, sex, mean arterial pressure, HbA1c, GFR, and the urine albumin-to-creatinine ratio (ACR). Baseline mean age (±SD) was 43±10 years, HbA1c 8.9±2.5%, GFR 147±46 ml/min, and median (IQR) ACR 41 (11-230) mg/g. During a median follow-up of 17.7 years, 70 participants developed kidney failure. After adjustment, each 1 SD increment in ωo was associated with a higher risk of kidney failure (HR=1.49, 95% CI 1.13, 1.96). Enhanced transglomerular passage of test macromolecules associated with progression to kidney failure, independent of albuminuria and GFR, suggesting that mechanisms associated with impaired glomerular permselectivity are important determinants of progressive kidney disease.

Early-onset gout and rare deficient variants of the lactate dehydrogenase D gene.

To investigate whether the lactate dehydrogenase D (LDHD) gene deficiency causes juvenile-onset gout. We used whole-exome sequencing for two families and a targeted gene-sequencing panel for an isolated patient. d-lactate dosages were analysed using ELISA. We demonstrated linkage of juvenile-onset gout to homozygous carriage of three rare distinct LDHD variants in three different ethnicities. In a Melanesian family, the variant was (NM_153486.3: c.206C>T; rs1035398551) and, as compared with non-homozygotes, homozygotes had higher hyperuricaemia (P = 0.02), lower fractional clearance of urate (P = 0.002), and higher levels of d-lactate in blood (P = 0.04) and urine (P = 0.06). In a second, Vietnamese, family, very severe juvenile-onset gout was linked to homozygote carriage of an undescribed LDHD variant (NM_153486.3: c.1363dupG) leading to a frameshift followed by a stop codon, p.(AlaGly432fsTer58). Finally, a Moroccan man, with early-onset and high d-lactaturia, whose family was unavailable for testing, was homozygous for another rare LDHD variant [NM_153486.3: c.752C>T, p.(Thr251Met)]. Rare, damaging LDHD variants can cause autosomal recessive early-onset gout, the diagnosis of which can be suspected by measuring high d-lactate levels in the blood and/or urine.

Purine metabolism state in patients with type 1 diabetes mellitus

The aim of the study. To carry out a comprehensive analysis of the purine metabolism (PM) state and assess its features in patients with type 1 diabetes mellitus (DM). Materials and methods. 181 patients with type 1 DM were examined (94 women and 87 men) aged 42.5 ± 12.1 years. Indicators of the carbohydrate metabolism state, creatinine (Cr) concentration by the method of Popper, purine bases (PBs) and the activity of xanthine oxidase (XO) by a photometric method, uric acid (UA) by a colorimetric method were determined in fasting venous blood. UA excretion was detected by the colorimetric method, Cr by an enzymatic method. Renal UA clearance (RCUA), fractional UA clearance (FCUA), total UA tubular reabsorption (TTRUA) and hypoxanthine-guanine-phosphoribosyltransferase (HGPRT) activity were calculated. The comparison group, representative in terms of age and sex, included 25 healthy volunteers. Results. PM changes in patients with type 1 DM were orientated towards an excessively increased catabolism and insufficient reutilization of PBs. The structure of the detected disorders was as follows: hyperuricemia (HU) (13.8 %), enhanced RCUA (42.8 %), increased XO activity (35.6 %) and inhibition of HGPRT activity (53.3 %). In about 56 % of the subjects, high concentrations of PBs were found, and HU was diagnosed only in every seventh subject. It was identified that PB concentrations were negatively correlated with the level of XO activity. RCUA and XO activity levels were revealed to be of the greatest informational value for assessing the PM state in patients with type 1 DM. RCUA was significantly associated with the level of UA excretion and HGPRT activity. Relationships of RCUA and FCUA with HbAc1 levels were established. The higher the level of HbAc1, the greater the clearance of RCUA was, especially FCUA, which led to a significant decrease in TTRUA. Conclusions. PM in type 1 DM is characterized by a high intensity, which is realized due to a decrease in anabolism, increased oxidation and suppression of PB reutilization. Uricemia inadequately reflects the level of UA production in patients with type 1 DM. The severity of PM disorders in type 1 DM patients is associated with the carbohydrate metabolism compensation state.

Relationship of apolipoprotein(a) isoform size with clearance and production of lipoprotein(a) in a diverse cohort

Lipoprotein(a) [Lp(a)] has two main proteins, apoB100 and apo(a). High levels of Lp(a) confer an increased risk for atherosclerotic cardiovascular disease. Most people have two circulating isoforms of apo(a) differing in their molecular mass, determined by the number of Kringle IV Type 2 repeats. Previous studies report a strong inverse relationship between Lp(a) levels and apo(a) isoform sizes. The roles of Lp(a) production and fractional clearance and how ancestry affects this relationship remain incompletely defined. We therefore examined the relationships of apo(a) size with Lp(a) levels and both apo(a) fractional clearance rates (FCR) and production rates (PR) in 32 individuals not on lipid-lowering treatment. We determined plasma Lp(a) levels and apo(a) isoform sizes, and used the relative expression of the two isoforms to calculate a “weighted isoform size” (wIS). Stable isotope studies were performed, using D3-leucine, to determine the apo(a) FCR and PR. As expected, plasma Lp(a) concentrations were inversely correlated with wIS (R2 = 0.27; P = 0.002). The wIS had a modest positive correlation with apo(a) FCR (R2 = 0.10, P = 0.08), and a negative correlation with apo(a) PR (R2 = 0.11; P = 0.06). The relationship between wIS and PR became significant when we controlled for self-reported race and ethnicity (SRRE) (R2 = 0.24, P = 0.03); controlling for SRRE did not affect the relationship between wIS and FCR. Apo(a) wIS plays a role in both FCR and PR; however, adjusting for SRRE strengthens the correlation between wIS and PR, suggesting an effect of ancestry.

New insights into proteinuria/albuminuria.

The fractional clearance of proteins as measured in healthy human subjects increases 10,000–100,000- fold when studied in nephrotic patients. This remarkable increase cannot be accounted for by extracellular biophysical mechanisms centered at the glomerular filtration barrier. Rather, it is the nephron and its combination of filtration and cellular uptake that can provide a plausible explanation of these fractional clearance changes. The nephron has two regions that critically determine the level proteinuria/albuminuria. Glomerular filtration of plasma proteins is primarily a size selective event that is basically unchanged in acquired and genetic kidney disease. The glomerular concepts of ‘charge selectivity’ and of ‘large pores’, previously used to explain proteinuria, are now recognized to be flawed and non-existent. Filtered proteins then encounter downstream two protein receptors of the Park and Maack type associated with the proximal tubular cell. The high capacity receptor is thought to retrieve the majority of filtered proteins and return them to the blood supply. Inhibition/saturation of this pathway in kidney disease may create the nephrotic condition and hypoproteinemia/hypoalbuminemia. Inhibitors of this pathway (possibly podocyte derived) are still to be identified. A relatively small proportion of the filtered protein is directed towards a high affinity, low capacity receptor that guides the protein to undergo lysosomal degradation. Proteinuria in normoproteinemic states is derived by inhibition of this pathway, such as in diabetes. The combination of glomerular sieving, and the degradation and retrieval pathways can quantitatively account for the changes in fractional clearance of proteins in the nephrotic condition. Finally, the general retrieval of filtered protein by the proximal tubular cell focuses on the teleological importance of this cell as this retrieval represents the third pillar of retrieval that this cell participates in (it also retrieves water and salt).

Serum Sclerostin Level Is Negatively Associated with Bone Mineral Density in Hemodialysis Patients.

Background and Objectives: Sclerostin and Dickkopf-1 (DKK1) modulate osteoblastogenesis, but their role in bone loss in hemodialysis (HD) patients is inconclusive. This study investigated relationships among lumbar bone mineral density (BMD), serum sclerostin, and DKK1 in HD patients. Materials and Methods: Blood samples were obtained from 75 HD patients. Dual-energy X-ray absorptiometry measured lumbar BMD of the lumbar vertebrae (L2–L4). Enzyme-linked immunosorbent assay revealed serum sclerostin and DKK1 concentrations. Results: There were 10 (13.3%), 20 (26.7%), and 45 (60%) patients defined as presenting with osteoporosis, osteopenia, or normal BMD, respectively. Age, alkaline phosphatase, urea reduction rate, fractional clearance index for urea, sclerostin level, and percentage of female patients are significantly negatively associated with the lumbar BMD and T-score, while the body mass index and waist circumference significantly positively associated with the lumbar BMD and T-score. Multivariate forward stepwise linear regression analysis indicated that serum sclerostin (β = −0.546, adjusted R2 change = 0.454; p < 0.001), age (β = −0.216, adjusted R2 change = 0.041; p = 0.007), and percentage of female HD patients (β = −0.288, adjusted R2 change = 0.072; p = 0.0018) were significantly negatively associated with lumbar BMD in HD patients. Conclusions: Advanced age, female gender, and serum sclerostin level, but not DKK1, were negatively associated with BMD in HD patients.

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.

This Month in Anesthesiology

This Month in Anesthesiology

Impaired Tubular Secretion of Organic Solutes in Advanced Chronic Kidney Disease.

The clearance of solutes removed by tubular secretion may be altered out of proportion to the GFR in CKD. Recent studies have described considerable variability in the secretory clearance of waste solutes relative to the GFR in patients with CKD. 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 patients with CKD and an eGFR of 7±2 ml/min per 1.73 m2 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 patients with CKD and to those of creatinine in controls. Metabolomic analysis identified 39 secreted solutes with fractional clearance >3.0 in control participants. Fractional clearance values in patients with CKD 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 patients with CKD; P<0.001), p-cresol sulfate (8.6±2.6 for controls versus 4.1±1.5 for patients with CKD; 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). Secretory clearances for many solutes are reduced more than the GFR in advanced CKD. Impaired secretion of these solutes might contribute to uremic symptoms as patients approach dialysis.

Анализ связи I/D полиморфизма гена ангиотензинпревращающего фермента с обменом мочевой кислоты у больных сахарным диабетом 2 типа (жителей Чувашии)

It is known that metabolic disorders of uric acid (UA), in particular hyperuricemia (HU), are associated with vascular complications of type 2 diabetes mellitus (DM 2). Previously, the authors established a relationship between the I/D polymorphism of the angiotensin-converting enzyme (ACE) gene and vascular lesions in patients with DM 2 living in Chuvashia. It was of interest to find out how the I/D polymorphism of the ACE gene and metabolic disorders of MC in patients with diabetes mellitus 2 (residents of Chuvashia) are related. ACE gene polymorphism was determined by the I/D polymerase reaction method in 104 patients with diabetes mellitus 2, in 46 of whom UA metabolism was studied in depth. The UA was determined spectrophotometrically in the blood and in daily urine, and the total and fractional renal clearance of the UA was calculated. There were no significant differences in the parameters of UA metabolism depending on the I/D polymorphism. A steady tendency towards a decrease in renal UA clearance was established in the DD genotype. In patients with the DD genotype in comparison with carriers of the I allele (genotypes ID and DD), a lower mean value of UA clearance was recorded (M 10.0, Me 8.4 ml/min versus M 12.4, Me 11.7 ml/min; Z=1.1, p=0.28) and a higher prevalence of its low (&lt;9 ml/min) values: 60% (9 out of 15) versus 34.6% (9 out of 26) (χ2=2, 18, p=0.14). It should be assumed that with an increase in the number of examined patients, the relationship of the DD genotype with a reduced UA clearance will acquire statistical significance. With the DD genotype, according to many authors, the maximum ACE activity is rectified. The authors suggest that an increase in ACE activity contributes to a decrease in UA transit through the kidneys in the DD genotype

Effects of PNPLA3 I148M on hepatic lipid and very-low-density lipoprotein metabolism in humans.

The phospholipase domain-containing 3 gene (PNPLA3)-148M variant is associated with liver steatosis but its influence on the metabolism of triglyceride-rich lipoproteins remains unclear. Here, we investigated the kinetics of large, triglyceride-rich very-low-density lipoprotein (VLDL), (VLDL1 ), and smaller VLDL2 in homozygotes for the PNPLA3-148M variant. The kinetics of apolipoprotein (apo) B100 (apoB100) and triglyceride in VLDL subfractions were analysed in nine subjects homozygous for PNPLA3-148M and nine subjects homozygous for PNPLA3-148I (controls). Liver fat was >3-fold higher in the 148M subjects. Production rates for apoB100 and triglyceride in VLDL1 did not differ significantly between the two groups. Likewise, production rates for VLDL2 -apoB100 and -triglyceride, and fractional clearance rates for both apoB100 and triglyceride in VLDL1 and VLDL2 , were not significantly different. Despite the higher liver fat content in PNPLA3 148M homozygotes, there was no increase in VLDL production. Equally, VLDL production was maintained at normal levels despite the putative impairment in cytosolic lipid hydrolysis in these subjects.

Abstract P182: Recombinant Human Lecithin-cholesterol Acyltransferase Treatment In Patients With ASCVD Increases HDL Through Increases In Cholesterol Ester Content.

Lecithin-cholesterol acyltransferase (LCAT) mediates the esterification of free cholesterol (FC) to cholesteryl ester (CE) in HDL and, therefore, plays a critical role in reverse cholesterol transport. MEDI6012, a recombinant human LCAT (rhLCAT) increases HDL-C. Our goal was to interrogate the pathways regulating the increase in HDL-C and effects of rhLCAT on apoB metabolism. Methods: We enrolled five subjects (4 Man, mean age 67) with stable ASCVD into a Phase II, placebo controlled, double-blind, randomized cross-over study to determine the effects of two IV doses of MEDI6012, administered 48-hrs apart, versus placebo, on plasma lipids and lipoproteins. Stable isotope kinetic studies with D2-Leu, 13C-Phe, D2-Glycerol were performed to examine the metabolism of apoB100, ApoA1, ApoA2 and triglyceride. Results: As expected two doses of IV MEDI6012 increased total cholesterol and HDL-C levels significantly (Table1). We did not observe significant changes in other measured lipids or lipoproteins. The significant increase in HDL-C was 34.9±10.3 (p=0.002) and due to an increase in the amount of CE (33±8.9 p=0.001); there was no change in free cholesterol (1.9±1.9). We found no changes in levels of sterols (i.e. Lathosterol) between the two periods. Preliminary in vivo kinetic studies of HDL metabolism in 3 subjects, showed no changes in the mean fractional clearance rate (FCR) or production rate (PR) of apoA1 between placebo (0.3±0.2pool/day, 1.3±0.5 mg/kg/day) and rhLCAT treatment (0.28±0.1pools/day, 1.3±0.3). There were no changes in ApoA2 FCR and PR after rhLCAT administration. Complete data for ApoA1, ApoA2 and ApoB100 and TG kinetics will be presented. Conclusions: In subjects with ASCVD, treatment with rhLCAT increased HDL-C mainly by increasing HDL CE; there were no changes in the FCR or PR of HDL ApoA1 or ApoA2. Together, these results suggest that rhLCAT treatment is associated with increased steady-state transport of CE by HDL.

Unraveling the Complexities of the Lipoprotein(a) Particle and its Metabolism

Lipoprotein (a) [Lp(a)], was discovered more than 60 years ago, however, its link to human disease has just begun to unfold. Recent findings in large genome association studies and Mendelian randomization studies have highlighted its role as a causal risk factor in coronary and atherogenic vascular disease. Despite years of research into the mechanisms regulating plasma levels of Lp(a), specific sites and pathways of regulation are poorly characterized. Lp(a) contains two main apolipoproteins, apolipoprotein (a) [apo(a)] and apolipoprotein B100 (apoB100), these are bound by covalent bounds but also weak lisine binding sites are present. The most significant determinant of the level of Lp(a) is the number of DNA-base repeats coding a highly polymorphic sequence of the LPA gene called Kringle IV Type 2 (KIV-2). Importantly, the number of KIV-2 repeats range from about 3-40 resulting in a range of apo(a) mass from 200 – 700 kDA. Almost all individuals, therefore, have two apo(a) isoforms that differ in the size and the size of the apoB bound to the apo(a) has not been well characterize. The mechanism(s) by which KIV-2 isoform size affect Lp(a) concentrations remain minimally defined. Previous studies, using isolated and exogenously radio-iodinated apo(a) isoforms, indicated that the hepatic secretion rate (SR) of apo(a), rather than its fractional clearance rate (FCR), determined plasma levels of Lp(a), and that smaller isoforms (fewer KIV-2 repeats) were associated with greater SRs and higher plasma levels of Lp(a). However isoform size does not account for all the variation in plasma Lp(a) concentration. Indeed, more recent studies using stable isotopes by our group and by others provide a much more complex picture. We will present data on recently completed mechanistic studies using stable isotope in human volunteers with high and low levels of Lp(a). The studies describe the clearance and production of apo(a) and apoB100 from isolated Lp(a). Additionally proteomic composition of the isolated Lp(a) particles and isoforms and the implications of these proteins on disease will be discussed. The studies presented will highlight the complexity of Lp(a) and provide novel insights into the regulation of this cardiovascular risk.