Fractional Turnover Research Articles

TRACING THE COORDINATED CONTROL OF NAD+ HOMEOSTASIS USING C. ELEGANS

Abstract Nicotinamide adenine dinucleotide (NAD+) is a vital coenzyme involved in redox reactions and cellular signaling. Its levels decline with age in humans, mice, and worms, leading to metabolic disturbances associated with age-related diseases. Interestingly, our studies in aged mice indicate that the rate of NAD+ synthesis remains unchanged, ruling out a lack of precursors as the cause of NAD+ decline. Instead, we found that tissues with lower NAD+ concentrations in aged mice exhibit higher fractional turnover, suggesting increased consumption as the primary driver of NAD+ decline during aging. This led us to hypothesize that the loss of NAD+ metabolic robustness and resiliency with age is linked to the hyper-activation and competition of NAD+-consuming enzymes. To investigate the physiological impact of NAD+ consuming enzymes on metabolic robustness, we used genetic tools and mass spectrometry in C. elegans. Surprisingly, we identified novel reproductive and aging phenotypes in mutant animals lacking specific NAD+ consuming enzymes, including Poly ADP-Ribose Polymerase (parp-1 and parp-2), TANKyrase (tank-1), and Toll and Interleukin 1 Receptor (tir-1). These mutants displayed significant reductions in fecundity and extensions in longevity. Additionally, employing High-Performance Liquid Chromatography coupled with Mass Spectrometry (HP-LC-MS), we examined the NAD+ metabolome across different developmental stages in our mutant strains, revealing notable metabolic disruptions associated with the loss of NAD+ consumers. In summary, our findings establish a novel connection between the hyper-activation of NAD+ degradation and the onset of reproductive aging. This research provides valuable insights into the role of NAD+ metabolism and its impact on aging-related processes.

Population pharmacokinetic-pharmacodynamic modeling of clopidogrel for dose regimen optimization based on CYP2C19 phenotypes: A proof of concept study.

Clopidogrel is an antiplatelet drug used to reduce the risk of acute coronary syndrome and stroke. It is converted by CYP2C19 to its active metabolite; therefore, poor metabolizers (PMs) of CYP2C19 exhibit diminished antiplatelet effects. Herein, we conducted a proof-of-concept study for using population pharmacokinetic-pharmacodynamic (PK-PD) modeling to recommend a personalized clopidogrel dosing regimen for individuals with varying CYP2C19 phenotypes and baseline P2Y12 reaction unit (PRU) levels. Data from a prospective phase I clinical trial involving 36 healthy male participants were used to develop the population PK-PD model predicting the concentrations of clopidogrel, clopidogrel H4, and clopidogrel carboxylic acid, and linking clopidogrel H4 concentrations to changes in PRU levels. A two-compartment model effectively described the PKs of both clopidogrel and clopidogrel carboxylic acid, and a one-compartment model of those of clopidogrel H4. The CYP2C19 phenotype was identified as a significant covariate influencing the metabolic conversion of the parent drug to its metabolites. A PD submodel of clopidogrel H4 that stimulated the fractional turnover rate of PRU levels showed the best performance. Monte Carlo simulations suggested that PMs require three to four times higher doses than extensive metabolizers to reach the target PRU level. Individuals within the top 20th percentile of baseline PRU levels were shown to require 2.5-3 times higher doses than those in the bottom 20th percentile. We successfully developed a population PK-PD model for clopidogrel considering the impact of CYP2C19 phenotypes and baseline PRU levels. Further studies are necessary to confirm actual dosing recommendations for clopidogrel.

Reduced Digestibility of Beef Tallow and Cocoa Butter Affects Bile Acid Excretion and Reduces Hepatic Esterified Cholesterol in Rats

We investigated stearic acid (18:0) digestibility and how it affects bile acid excretion in male Sprague-Dawley rats fed diets containing (g 18:0/100 g fatty acids): pork lard (13); beef tallow (19); cocoa butter (35); corn oil (2) or corn oil plus cholestyramine for 25 d. Apparent lipid digestibility was reduced with increased dietary intake of 18:0 as follows: lard (90%), beef tallow (82%), cocoa butter (78%), cholestyramine (87%), and corn oil (94%); P < 0.001, pooled SD = 2. Hepatic concentrations of total and esterified cholesterol were significantly less in cocoa butter-, beef tallow- and cholestyramine-fed groups compared with lard- and corn oil-fed groups. Fecal bile acid excretion was significantly greater in rats fed cocoa butter or cholestyramine compared with those fed corn oil. The half-life of intraperitoneally administered 14C-cholic acid was significantly longer in rats fed cocoa butter (1.36 ± 0.02 d) compared with cholestyramine (0.98 ± 0.03 d) and intermediate in those fed corn oil, lard or beef tallow (1.11–1.21 ± 0.05 d). Fecal excretion of muricholic acids (bile acids) correlated strongly with dietary intake of 18:0 (r2 = 0.98, P < 0.01), whereas excretion of bile acids derived from cholic and chenodeoxycholic acids was similar among groups. In summary, the lower digestibility of cocoa butter is associated with increased fecal bile acid excretion, reduced hepatic concentration of esterified cholesterol, decreased fractional turnover of 14C-cholic acid and increased excretion of muricholic acids in rats. The mechanism by which stearate-rich dietary fats alter bile acid and cholesterol metabolism is, however, uncertain.

918-P: Predicting the Onset of APECED Diabetes

Background: Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a rare monogenic autosomal recessive disorder and diabetes (DM) one of its rare and less studied manifestations. Determinants of time to onset and clinical progression to DM seem currently unpredictable. Materials and Methods: We explored change in pre-diagnosis (dg) glycemic parameters (glucose, insulin, C-peptide), including 60’ intravenous glucose tolerance tests (ivGTT) and homeostasis indices (HOMA-β, -IR) over 20 years (y) with descriptive statistics. Fractional glucose turnover rate (KG) during ivGTT was calculated as 0.693/T1/2Gluc*100 with Pearson correlation coefficient (r) and 2-sided p value. Results: Between 1972 and 1996, 68 ivGTTs were conducted in 13 pediatric and adult individuals with APECED and DM. The mean age (SD) at DM dg was 27.78y (11.73) among those 22% of our cohort (n=100) with DM. Including one man with ‘latent DM’, with no clinical DM symptoms 20y post-dg. HbA1c was rarely measured, fasting plasma glucose (FPG) values were normoglycemic prior and up to DM dg: mean (SD) FPG 4.4 (0.6) mmol/l (-20y to -6 months [m]) and 5.7 (0.9) mmol/l (&amp;lt;6m to dg). Mean 60’ glucose values were only elevated for those with &amp;lt;6m to dg: 12.1 (0.7) mmol/l. Hyperinsulinemia was present 5-20y prior to dg while rapid 1st phase insulin secretion (combined 1+3 min value; normal &amp;gt;46 mIU/ml) was lost or delayed in some, but not all, 5y prior to dg. Up to and at dg, adequate fasting insulin (&amp;gt;2 mIU/ml) and C-peptide (&amp;gt;0.5 ng/ml) production was observed in most (85%) patients. KG displayed intra- and inter-individual variability, was normal (&amp;gt;1.2%/min) in latent DM, while it predicted DM onset over time (-20y to dg): r= -0.568 (p= 4E-06). Neither HOMA indices were sensitive enough to detect any change over time. Conclusion: Identification of reduced glucose removal rate over time, indicative of increased peripheral insulin resistance in the presence of pre-diagnostic hyperinsulinemia, provides first insights into progression of atypical diabetes in APECED patients. Disclosure P. M. Paldanius: Consultant; Self; UPM Biomedicals, Speaker’s Bureau; Self; Novartis AG. O. Makitie: None. S. Laakso: None.

Methane dynamics in three different Siberian water bodies under winter and summer conditions

Abstract. Arctic regions and their water bodies are affected by a rapidly warming climate. Arctic lakes and small ponds are known to act as an important source of atmospheric methane. However, not much is known about other types of water bodies in permafrost regions, which include major rivers and coastal bays as a transition type between freshwater and marine environments. We monitored dissolved methane concentrations in three different water bodies (Lena River, Tiksi Bay, and Lake Golzovoye, Siberia, Russia) over a period of 2 years. Sampling was carried out under ice cover (April) and in open water (July–August). The methane oxidation (MOX) rate and the fractional turnover rate (k′) in water and melted ice samples from the late winter of 2017 was determined with the radiotracer method. In the Lena River winter methane concentrations were a quarter of the summer concentrations (8 nmol L−1 vs. 31 nmol L−1), and mean winter MOX rate was low (0.023 nmol L−1 d−1). In contrast, Tiksi Bay winter methane concentrations were 10 times higher than in summer (103 nmol L−1 vs. 13 nmol L−1). Winter MOX rates showed a median of 0.305 nmol L−1 d−1. In Lake Golzovoye, median methane concentrations in winter were 40 times higher than in summer (1957 nmol L−1 vs. 49 nmol L−1). However, MOX was much higher in the lake (2.95 nmol L−1 d−1) than in either the river or bay. The temperature had a strong influence on the MOX (Q10=2.72±0.69). In summer water temperatures ranged from 7–14 ∘C and in winter from −0.7 to 1.3 ∘C. In the ice cores a median methane concentration of 9 nM was observed, with no gradient between the ice surface and the bottom layer at the ice–water interface. MOX in the (melted) ice cores was mostly below the detection limit. Comparing methane concentrations in the ice with the underlaying water column revealed methane concentration in the water column 100–1000 times higher. The winter situation seemed to favor a methane accumulation under ice, especially in the lake with a stagnant water body. While on the other hand, in the Lena River with its flowing water, no methane accumulation under ice was observed. In a changing, warming Arctic, a shorter ice cover period is predicted. With respect to our study this would imply a shortened time for methane to accumulate below the ice and a shorter time for the less efficient winter MOX. Especially for lakes, an extended time of ice-free conditions could reduce the methane flux from the Arctic water bodies.

An Integrative Approach for Improved Assessment of Cardiovascular Safety Data.

Cardiovascular adverse effects in drug development are a major source of compound attrition. Characterization of blood pressure (BP), heart rate (HR), stroke volume (SV), and QT-interval prolongation are therefore necessary in early discovery. It is, however, common practice to analyze these effects independently of each other. High-resolution time courses are collected via telemetric techniques, but only low-resolution data are analyzed and reported. This ignores codependencies among responses (HR, BP, SV, and QT-interval) and separation of system (turnover properties) and drug-specific properties (potencies, efficacies). An analysis of drug exposure-time and high-resolution response-time data of HR and mean arterial blood pressure was performed after acute oral dosing of ivabradine, sildenafil, dofetilide, and pimobendan in Han-Wistar rats. All data were modeled jointly, including different compounds and exposure and response time courses, using a nonlinear mixed-effects approach. Estimated fractional turnover rates [h-1, relative standard error (%RSE) within parentheses] were 9.45 (15), 30.7 (7.8), 3.8 (13), and 0.115 (1.7) for QT, HR, total peripheral resistance, and SV, respectively. Potencies (nM, %RSE within parentheses) were IC 50 = 475 (11), IC 50 = 4.01 (5.4), EC 50 = 50.6 (93), and IC 50 = 47.8 (16), and efficacies (%RSE within parentheses) were I max = 0.944 (1.7), Imax = 1.00 (1.3), E max = 0.195 (9.9), and Imax = 0.745 (4.6) for ivabradine, sildenafil, dofetilide, and pimobendan. Hill parameters were estimated with good precision and below unity, indicating a shallow concentration-response relationship. An equilibrium concentration-biomarker response relationship was predicted and displayed graphically. This analysis demonstrates the utility of a model-based approach integrating data from different studies and compounds for refined preclinical safety margin assessment. SIGNIFICANCE STATEMENT: A model-based approach was proposed utilizing biomarker data on heart rate, blood pressure, and QT-interval. A pharmacodynamic model was developed to improve assessment of high-resolution telemetric cardiovascular safety data driven by different drugs (ivabradine, sildenafil, dofetilide, and pimobondan), wherein system- (turnover rates) and drug-specific parameters (e.g., potencies and efficacies) were sought. The model-predicted equilibrium concentration-biomarker response relationships and was used for safety assessment (predictions of 20% effective concentration, for example) of heart rate, blood pressure, and QT-interval.

Fractional turnover of apolipoprotein(a) and apolipoprotein B-100 within plasma lipoprotein(a) particles in statin-treated patients with elevated and normal Lp(a) concentration

ContextLipoprotein(a) [Lp(a)] is a highly atherogenic lipoprotein characterized by apolipoprotein(a) [apo(a)] covalently bounded to apoB-100 (apoB). However, the metabolism of apo(a) and apoB within plasma Lp(a) particles in patients on statins remains unclear. MethodsThe kinetics of Lp(a)-apo(a) and Lp(a)-apoB were determined in 20 patients with elevated Lp(a) (≥0.8 g/L; n = 10) and normal Lp(a) (≤0.3 g/L; n = 10) using stable isotope techniques and compartmental modeling. Plasma apo(a) concentration was measured using liquid chromatography–mass spectrometry. All patients were on statin therapy and were studied in the fasting state. ResultsThe fractional catabolic rate (FCR) of Lp(a)-apo(a) was not significantly different from that of Lp(a)-apoB in statin-treated patients with elevated or normal Lp(a) (P > 0.05 in both). Lp(a)-apo(a) FCR was significantly correlated with Lp(a)-apoB in patients with elevated and normal Lp(a) concentrations (r = 0.970 and r = 0.979, respectively; all P < 0.001) with Lin's concordance test showing substantial agreement between the FCRs of Lp(a)-apo(a) and Lp(a)-apoB in patients with elevated and normal Lp(a) concentrations (rc = 0.978 and rc = 0.966, respectively). ConclusionOur data indicate that the apo(a) and apoB proteins within Lp(a) particles have similar FCR and are therefore tightly coupled as an Lp(a) holoparticle in statin-treated patients with elevated and normal Lp(a) concentrations.

Afforestation of loess soils: Old and new organic carbon in aggregates and density fractions

Afforestation is an important strategy to increase soil organic carbon (SOC) stocks and stabilize soils against degradation and erosion. While the area under afforestation has increased globally during the last 20 years, the dynamics of SOC accumulation due to afforestation are still poorly known, as it cannot be directly compared to that in cropland. The goals of this study were: i) to investigate the dynamics of SOC accumulation after afforestation on the Loess Plateau; ii) to determine SOC contents and distribution in density fractions and aggregates most strongly affected by afforestation; and iii) to analyze the relationships between old and new SOC in relation with afforestation periods. A chronosequence of forest plots aged 1, 5, 12 (with Chinese pine, Pinus tabulaeformis Carr.) and 30 years (with white poplar, Populus alba L.) were selected within the large scale “Grain for Green” project. A maize field nearby was chosen as a control, which represents the land use prior to afforestation. At each location, soils were sampled at depths of 0–10 and 10–30 cm, respectively and aggregates were fractionated into <250, 250–2000 and > 2000 μm sizes, which were subsequently separated into light (ρ < 1.85 g cm−3) and heavy (ρ > 1.85 g cm−3) density fractions. SOC content was generally higher in afforested soils than in cropland and increased with stand age. The mean SOC accumulation rate was 0.11 g C kg−1 year−1 for the 0–10 cm layer of the 30-year-old forests, whereas the maximum rate was found for 5-year-old forest at 0.24 g C kg−1 year−1. For each period of afforestation, the maximum SOC content was recorded in the macroaggregates. The C turnover of the light fraction (calculated based on δ13C of SOC) peaked in the large macroaggregates and ranged from 21 to 23 years). Whereas SOC turnover in the heavy fraction, peaked in the microaggregates at a relatively longer period of 46 to 70 years. We conclude that the initial SOC accumulation under afforestation occurs mainly in the macroaggregates, with a faster turnover compared with microaggregates. During the first 30 years of afforestation, the SOC accumulation and stabilization is ongoing mainly in the upper 10 cm, while the C sequestration in 10 to 30 cm depth needs much longer time.

Plasma Dexamethasone Concentration in Relation to Glucose Response in the Horse

Therapeutic agents capable of altering the performance of horses are monitored in racing and equestrian sports to guarantee horse welfare, fair competition, and integrity of the sport. Dexamethasone is a common glucocorticoid drug for treating horses. Among other effects, dexamethasone is gluconeogenic and increases blood glucose. In this study, plasma samples from two dexamethasone exposure studies were analyzed for glucose using an automated clinical chemistry analyzer. In study one, dexamethasone-21-isonicotinate was administered to six horses at the dose 30 μg/kg intramuscularly. In study two, dexamethasone 21-phosphate disodium salt was administered intravenously to six horses as a bolus dose followed by 3 hours of infusion (bolus + infusion) at four different doses (placebo, 0.1 + 0.07 μg/kg, 1 + 0.7 μg/kg, and 10+7 μg/kg). Plasma dexamethasone concentrations were linked to plasma glucose concentrations by means of a turnover model. The median (range) pharmacodynamic parameters for glucose response for the two studies were as follows: the EC50 value was 0.84 μg/L (0.47–1.50) and 0.85 μg/L (0.75–2.45), the fractional turnover rate of response was 0.18 per h (0.07–0.27) and 0.25 per h (0.17.0.48), and the unaffected baseline of response was 4.20 mmol/L (4.10–6.60) and 5.42 mmol/L (5.22–5.96). These results may be used as input to future studies of the anti-inflammatory response of dexamethasone. The results also enable calculations of irrelevant plasma concentrations to determine whether the presence of a drug is a trace from legitimate medication or not. Therefore, this study provides further evidence for dexamethasone screening limits, which protects the integrity of the sport and the welfare of the horse.

SGLT Inhibitors Enhance Endogenous Glucose Productions (EGP) in a Preclinical Animal Model

Aim: SGLT inhibitors was found to paradoxically enhance EGP in type 2 diabetes patients, while reduce EGP in diabetic mice during hyperinsulinemic-euglycemic clamp. In this study, we assess acute and 7-day effects of SGLT inhibitors on EGP in rats by 13C-glucose tracer dilution method to identify a translational rodent model. Method: SD rats aged 7 weeks were assigned to 3 groups (n=6 each), vehicle, LX4211 (10 mg/kg) and canagliflozin (10 mg/kg). On day 1, rats were fasted for 3 hours and dosed with SGLT inhibitors, LX4211 or canagliflozin before a 13C-glucose IV injection (25 mg/kg) 2 hour post drug administration. Blood were collected at 0, 5, 15, 30, 45, 60, 75, 90, 120, and 150 minutes time points. Plasma 13C-glucose and 12C-glucose levels were detected by LC/MS. EGP was calculated by multiplying fractional glucose turnover rate by pool size. After washout and the same 18 rats were dosed for 7 days, EGP were assessed on day 7. Results: 13C--glucose IV injection after acute LX4211 and canagliflozin dosing did not significantly changed plasma glucose concentration over time, but led to a rapid rise in plasma 13C-glucose enrichment followed by a time-dependent decrease of 13C-glucose enrichment. The fractional glucose turnover rate and EGP were 3.14±0.35 (SD) percent and 6.28±0.70 (SD) mg.kg-1.minutes-1 in vehicle group. Single dose of LX4211 and canagliflozin significantly increased EGP by 27.5% and 57.1%, respectively. After 7-days of treatment, the fractional glucose turnover and EGP were 3.30±0.28 (SD) percent and 6.59±0.56 (SD) mg.kg-1.minutes-1 in vehicle group, which was highly consistent to the previous EGP measurement. Moreover, 7 days treatment of LX4211 and canagliflozin significantly elevated EGP by 24.8% and 33.4%, respectively. Conclusion: This study demonstrated that SGLT inhibitors can increase EGP in animal model as in type 2 diabetes patients. Detecting EGP using 13C-glucose tracer dilution method may facilitate the discovery of novel SGLT inhibitors with differential effects on EGP. Disclosure S. Huang: None. D. Cai: None. Z. Yue: None. H. Yan: None. Z. Zhou: None. X. Fu: None. H. Wang: None. Y. Tao: None. J.J. Hwa: None.

Population pharmacodynamics of cilostazol in healthy Korean subjects.

Cilostazol is used for the treatment of intermittent claudication, ulceration and pain. This study was conducted to develop a population pharmacodynamic (PD) model for cilostazol's closure time (CT) prolongation effect in healthy Korean subjects based on a pharmacokinetic (PK) model previously developed. PD data were obtained from 29 healthy subjects who participated in a study conducted in 2009 at Severance Hospital. The PK model used was a two-compartment model with first order absorption. CT data were best described by a turnover model with a fractional turnover rate constant (Kout) inhibited by drug effects (Eff), which were represented by a sigmoid Emax model [Eff = Emax · Cγ / (EC50γ+Cγ)] with Emax being maximum drug effect, EC50 drug plasma concentration at 50% of Emax, C drug plasma concentrations, and γ the Hill coefficient. For the selected PD model, parameter estimates were 0.613 hr−1 for Kout, 0.192 for Emax, 730 ng/ml for EC50 and 5.137 for γ. Sex and caffeine drinking status significantly influenced the baseline CT, which was 85.36 seconds in male non-caffeine drinkers and increased by 15.5% and 16.4% in females and caffeine drinkers, respectively. The model adequately described the time course of CT. This was the first population PD study for cilostazol's CT prolongation effect in a Korean population.

Quantitative analysis of the effect of end-tidal carbon dioxide on regional cerebral oxygen saturation in patients undergoing carotid endarterectomy under general anaesthesia.

Regional cerebral oxygen saturation (rSO2 ) is currently the most used measure in clinical practice to monitor cerebral ischaemia in patients undergoing carotid endarterectomy (CEA). Although end-tidal carbon dioxide (PET CO2 ) is known as a factor that influences rSO2 , the relationship between PET CO2 and rSO2 has not been quantitatively evaluated in patients with severe arteriosclerosis. This study aimed to evaluate the effect of PET CO2 on rSO2 in patients undergoing CEA under general anaesthesia. The intervention to change PET CO2 was conducted between skin incision and clamping of the carotid artery. The rSO2 values were observed by changing PET CO2 in the range of 25-45mmHg. The PET CO2 -rSO2 relationship was characterized by population analysis using a turnover model. In total, 1651 rSO2 data points from 30 patients were used to determine the pharmacodynamic characteristics. Hypertension (HTN) and systolic blood pressure (SBP) were significant covariates on the slope factor in the stimulatory effect of PET CO2 on rSO2 and fractional turnover rate constant (kout ), respectively. The estimates of the parameters were kout (min-1 ): 3.59 for SBP<90mmHg and 0.491 for SBP ≥90mmHg, slope: 0.00321 for patients with HTN and 0.00664 for patients without HTN. The presence of HTNattenuates the response of rSO2 after a change in PET CO2 . When cerebral blood flow is in a state of decline caused by a decrease in SBP to <90mmHg, the response of rSO2 to PET CO2 is increased. It is advisable to maintain SBP >90mmHg in patients with HTNduring CEA.

Mechanistic Model for Blood Pressure and Heart Rate Changes Produced by Telmisartan in Human Beings.

Telmisartan, an angiotensin receptor blocker (ARB), is indicated for the treatment of essential hypertension. This study aimed to develop a mechanistic model of telmisartan drug effect in human beings using non-invasive markers. Data were acquired from a previous study where telmisartan 80mg was given once daily for 6days. Systolic (SBP) and diastolic blood pressure (DBP) and heart rate (HR) were measured before dosing for days 1-5 and serially after the last dose. Mean arterial pressure (MAP) and pulse pressure (PP) were calculated from SBP and DBP. Relationships between MAP, PP, HR and total peripheral resistance (TPR) were developed. Circadian variation was incorporated into PP and HR, and TPR was assumed to adjust itself in response to changes in PP and HR based on baroreflex mechanism. Drug effects were then described as lowering the set point of MAP through TPR with a physiological feedback effect stimulating HR and PP. Drug concentrations were described by a two-compartment disposition model with first-order absorption and lag time, and first-order elimination. Circadian variation was described by cosine functions, having periods of 12 and 24hr. A log-linear model was used to describe drug effect, with estimated drug effect parameter of 0.051/hr. Estimated fractional turnover rate of PP, HR and TPR was 11.2hr. The model successfully described the time courses of these cardiovascular variables. This work demonstrated the feasibility of using non-invasive cardiovascular measurements to derive a mechanistic model for telmisartan in human beings. The model may be suitable for other ARBs.

Enhancing Studies of Pharmacodynamic Mechanisms via Measurements of Metabolic Flux: Fundamental Concepts and Guiding Principles for Using Stable Isotope Tracers.

Drug discovery and development efforts are largely based around a common expectation, namely, that direct or indirect action on a cellular process (e.g., statin-mediated enzyme inhibition or insulin-stimulated receptor activation) will have a beneficial impact on physiologic homeostasis. To expand on this, one could argue that virtually all pharmacologic interventions attempt to influence the flow of "traffic" in a biochemical network, irrespective of disease or modality. Since stable isotope tracer kinetic methods provide a measure of traffic flow (i.e., metabolic flux), their inclusion in study designs can yield novel information regarding pathway biology; the application of such methods requires the integration of knowledge in physiology, analytical chemistry, and mathematical modeling. Herein, we review the fundamental concepts that surround the use of tracer kinetics, define basic terms, and outline guiding principles via theoretical and experimental problems. Specifically, one needs to 1) recognize the types of biochemical events that change isotopic enrichments, 2) appreciate the distinction between fractional turnover and flux rate, and 3) be aware of the subtle differences between tracer kinetics and pharmacokinetics. We hope investigators can use the framework presented here to develop applications that address their specific questions surrounding biochemical flux, and thereby gain insight into the pathophysiology of disease states, and examine pharmacodynamic mechanisms.

Amyloid β concentrations and stable isotope labeling kinetics of human plasma specific to central nervous system amyloidosis

IntroductionCerebrospinal fluid analysis and other measurements of amyloidosis, such as amyloid-binding positron emission tomography studies, are limited by cost and availability. There is a need for a more practical amyloid β (Aβ) biomarker for central nervous system amyloid deposition. MethodsWe adapted our previously reported stable isotope labeling kinetics protocol to analyze the turnover kinetics and concentrations of Aβ38, Aβ40, and Aβ42 in human plasma. ResultsAβ isoforms have a half-life of approximately 3 hours in plasma. Aβ38 demonstrated faster turnover kinetics compared with Aβ40 and Aβ42. Faster fractional turnover of Aβ42 relative to Aβ40 and lower Aβ42 and Aβ42/Aβ40 concentrations in amyloid-positive participants were observed. DiscussionBlood plasma Aβ42 shows similar amyloid-associated alterations as we have previously reported in cerebrospinal fluid, suggesting a blood-brain transportation mechanism of Aβ. The stability and sensitivity of plasma Aβ measurements suggest this may be a useful screening test for central nervous system amyloidosis.

Turnover of the Soil Organic Matter Amino Acid Fraction Investigated by13C and14C Signatures of Carboxyl Carbon

AbstractNitrogenous compounds of soil organic matter constitute a major N reservoir on Earth. Both the world food protein supply produced by agriculture and the global contamination by reactive nitrogen species rely on the dynamics of these compounds. To investigate their dynamics, we used both natural13C labeling and accelerator mass spectrometry (AMS)14C dating of the α-carboxyl amino carbon, which is specific of the amino acid fraction that was extracted from bulk soil organic matter by ninhydrin hydrolysis. We applied this isotopic approach to investigate the age of carboxyl carbon in a maize-cultivated Cambisol chronosequence. Based on a few measurements, we demonstrate the feasibility of this new compound-specific method of investigation of soil carbon dynamics. We show that soil organic matter amino acids can be split into two very distinct dynamic compartments: the majority having a mean age of a few years and a minority having a mean carbon age of several millennia. The latter fraction can be either strongly stabilized in soils, or can arise from microbial utilization of old carbon resources, as predicted by the priming effect theory.

Quantifying rates of glucose production in vivo following an intraperitoneal tracer bolus.

Aberrant regulation of glucose production makes a critical contribution to the impaired glycemic control that is observed in type 2 diabetes. Although isotopic tracer methods have proven to be informative in quantifying the magnitude of such alterations, it is presumed that one must rely on venous access to administer glucose tracers which therein presents obstacles for the routine application of tracer methods in rodent models. Since intraperitoneal injections are readily used to deliver glucose challenges and/or dose potential therapeutics, we hypothesized that this route could also be used to administer a glucose tracer. The ability to then reliably estimate glucose flux would require attention toward setting a schedule for collecting samples and choosing a distribution volume. For example, glucose production can be calculated by multiplying the fractional turnover rate by the pool size. We have taken a step-wise approach to examine the potential of using an intraperitoneal tracer administration in rat and mouse models. First, we compared the kinetics of [U-13C]glucose following either an intravenous or an intraperitoneal injection. Second, we tested whether the intraperitoneal method could detect a pharmacological manipulation of glucose production. Finally, we contrasted a potential application of the intraperitoneal method against the glucose-insulin clamp. We conclude that it is possible to 1) quantify glucose production using an intraperitoneal injection of tracer and 2) derive a "glucose production index" by coupling estimates of basal glucose production with measurements of fasting insulin concentration; this yields a proxy for clamp-derived assessments of insulin sensitivity of endogenous production.

Proteome Dynamics Reveals Pro-Inflammatory Remodeling of Plasma Proteome in a Mouse Model of NAFLD.

Nonalcoholic fatty liver disease (NAFLD) is associated with an increased risk of cardiovascular disease. Because the liver is the major source of circulatory proteins, it is not surprising that hepatic disease could lead to alterations in the plasma proteome, which are therein implicated in atherosclerosis. The current study used low-density lipoprotein receptor-deficient (LDLR(-/-)) mice to examine the impact of Western diet (WD)-induced NAFLD on plasma proteome homeostasis. Using a (2)H2O-metabolic labeling method, we found that a WD led to a proinflammatory distribution of circulatory proteins analyzed in apoB-depleted plasma, which was attributed to an increased production. The fractional turnover rates of short-lived proteins that are implicated in stress-response, lipid metabolism, and transport functions were significantly increased with WD (P < 0.05). Pathway analyses revealed that alterations in plasma proteome dynamics were related to the suppression of hepatic PPARα, which was confirmed based on reduced gene and protein expression of PPARα in mice fed a WD. These changes were associated with ∼4-fold increase (P < 0.0001) in the proinflammatory property of apoB-depleted plasma. In conclusion, the proteome dynamics method reveals proinflammatory remodeling of the plasma proteome relevant to liver disease. The approach used herein may provide a useful metric of in vivo liver function and better enable studies of novel therapies surrounding NAFLD and other diseases.

Microbial turnover of above and belowground litter components in shrublands

Shrublands cover a large proportion of the world’s land surface, yet they remain poorly studied in comparison to other ecosystems. Within shrublands, soil organic matter (SOM) is replenished from inputs of both above- and below-ground plant litter, however, their relative importance depends on their respective turnover rates. To critically address this, we measured the biodegradation rates of the soluble and insoluble components of 14C-labelled above- and below-ground plant litter in soil. During the 150day incubation, the amount of plant-derived soluble-C lost as 14CO2 was similar for the different plant parts being 64.7±2.3% for roots, 72.1±7.4% for stems, and 72.4±1.8% for leaves. In comparison, the turnover of the insoluble fraction was much slower. However, again little difference in mineralisation was seen for the different plant parts with the total losses being 21.1±0.9% for roots, 19.5±1.6% for stems, and 19.6±1% for leaves. A double exponential first order kinetic model fitted well to the experimental data. It also allowed the partitioning of C between microbial anabolic and catabolic processes for the soluble C component. Using this model, we deduced that the soluble fraction turns over ca. 40 times annually, whereas it takes ca. 2.5 years to turnover the insoluble fraction. For the soluble plant component, the overall microbial carbon use efficiency (CUE) was estimated to be greater for root-derived C in comparison to that derived from aboveground (no difference was observed for the insoluble component). From this, we tentatively suggest that C sourced from belowground plant components may persist longer in soil than C derived from aboveground plant components.

Precision-feeding dairy heifers a high rumen-degradable protein diet with different proportions of dietary fiber and forage-to-concentrate ratios

The objective of this experiment was to determine the effects of feeding a high-rumen-degradable protein (RDP) diet when dietary fiber content is manipulated within differing forage-to-concentrate ratio (F:C) on nutrient utilization of precision-fed dairy heifers. Six cannulated Holstein heifers (486.98±15.07kg of body weight) were randomly assigned to 2 F:C, low- (45% forage; LF) and high-forage (90% forage; HF) diets and to a fiber proportion sequence [33% grass hay and wheat straw (HS), 67% corn silage (CS; low fiber); 50% HS, 50% CS (medium fiber); and 67% HS, 33% CS (high fiber)] within forage proportion administered according to a split-plot, 3×3 Latin square design (16-d periods). Heifers fed LF had greater apparent total-tract organic matter digestibility coefficients (dC), neutral detergent fiber, and cellulose than those fed LC diets. Substituting CS with HS resulted in a linear reduction in dry matter, organic matter, and cellulose dC. Nitrogen dC was not different between F:C or with increasing proportions of HS in diets, but N retention tended to decrease linearly as HS was increased in the diets. Predicted microbial protein flow to the duodenum decreased linearly with HS addition and protozoa numbers HS interacted linearly, exhibiting a decrease as HS increased for LF, whereas no effects were observed for HF. Blood urea N increased linearly as HS was incorporated. The LF-fed heifers had a greater ruminal volatile fatty acids concentration. We noted a tendency for a greater dry matter, and a significantly higher liquid fraction turnover rate for HF diets. There was a linear numerical increase in the liquid and solid fraction turnover rate as fiber was added to the diets. Rumen fermentation parameters and fractional passages (solid and liquid) rates support the reduction in dC, N retention, and microbial protein synthesis observed as more dietary fiber is added to the rations of dairy heifers precision-fed a constant proportion of rumen-degradable protein.