Recombinant Adenosine Deaminase Ameliorates Inflammation, Vascular Disease, and Fibrosis in Preclinical Models of Systemic Sclerosis.

OBJECTIVE: Systemic sclerosis (SSc) is characterized by fibrosis, vascular disease, and inflammation. Adenosine signaling plays a central role in fibroblast activation. We undertook this study to evaluate the therapeutic effects of adenosine depletion with PEGylated adenosine deaminase (PEG-ADA) in preclinical models of SSc. METHODS: The effects of PEG-ADA on inflammation, vascular remodeling, and tissue fibrosis were analyzed in Fra-2 mice and in a B10.D2→BALB/c (H-2d ) model of sclerodermatous chronic graft-versus-host disease (GVHD). The effects of PEG-ADA were confirmed in vitro in a human full-thickness skin model. RESULTS: PEG-ADA effectively inhibited myofibroblast differentiation and reduced pulmonary fibrosis by 34.3% (with decreased collagen expression) (P = 0.0079; n = 6), dermal fibrosis by 51.8% (P = 0.0006; n = 6), and intestinal fibrosis by 17.7% (P = 0.0228; n = 6) in Fra-2 mice. Antifibrotic effects of PEG-ADA were also demonstrated in sclerodermatous chronic GVHD (reduced by 38.4%) (P = 0.0063; n = 8), and in a human full-thickness skin model. PEG-ADA treatment decreased inflammation and corrected the M2/Th2/group 2 innate lymphoid cell 2 bias. Moreover, PEG-ADA inhibited proliferation of pulmonary vascular smooth muscle cells (reduced by 40.5%) (P < 0.0001; n = 6), and prevented thickening of the vessel walls (reduced by 39.6%) (P = 0.0028; n = 6) and occlusions of pulmonary arteries (reduced by 63.9%) (P = 0.0147; n = 6). Treatment with PEG-ADA inhibited apoptosis of microvascular endothelial cells (reduced by 65.4%) (P = 0.0001; n = 6) and blunted the capillary rarefication (reduced by 32.5%) (P = 0.0199; n = 6). RNA sequencing demonstrated that treatment with PEG-ADA normalized multiple pathways related to fibrosis, vasculopathy, and inflammation in Fra-2 mice. CONCLUSION: Treatment with PEG-ADA ameliorates the 3 cardinal features of SSc in pharmacologically relevant and well-tolerated doses. These findings may have direct translational implications, as PEG-ADA has already been approved by the Food and Drug Administration for the treatment of patients with ADA-deficient severe combined immunodeficiency disease.

Determination of the reference range of endogenous plasma carnitines in healthy adults.

BACKGROUND: l-carnitine is an endogenous substance, vital in the transport of fatty acids across the inner mitochondrial membrane for oxidation. Disturbances in carnitine homeostasis can have a significant impact on human health; therefore, it is critical to define normal endogenous concentrations for l-carnitine and its esters to facilitate the diagnosis of carnitine deficiency disorders. This study was conducted to determine the normal concentrations of a number of carnitines in healthy adults using three analytical methods. The impact of age and gender on carnitine concentrations was also examined. METHODS: Blood samples were collected from 60 healthy subjects of both genders and various ages. Plasma samples were analysed for endogenous carnitine concentrations by radioenzymatic assay, high-performance liquid chromatography and electrospray tandem mass spectrometry. RESULTS: Precision and accuracy of results obtained for each assay were within acceptable limits. Average endogenous concentrations obtained from the three analytical methods in this study were in the range of 38-44, 6-7 and 49-50 mumol/L for l-carnitine, acetyl-l-carnitine and total carnitine, respectively. Comparison of results between the genders indicated that males had significantly higher endogenous plasma l-carnitine and total carnitine concentrations than females. Age was found to have no impact on plasma carnitine concentrations. CONCLUSION: These results are useful in the evaluation of biochemical or metabolic disturbances and in the diagnosis and treatment of patients with carnitine deficiency.

A pharmacokinetic model for L-carnitine in patients receiving haemodialysis.

AIMS: Patients requiring chronic haemodialysis may develop a secondary carnitine deficiency through dialytic loss of L-carnitine. A previous report has described the plasma concentrations of L-carnitine in 12 such patients under baseline conditions and after L-carnitine administration (20 mg kg(-1)). A three-compartment pharmacokinetic model was developed to describe these data to make inferences about carnitine supplementation in these patients. METHODS: L-carnitine removal was mediated solely by intermittent haemodialysis, which was incorporated into the model as an experimentally derived dialysis clearance value that was linked to an on-off pulse function. Data were described by a model with a central compartment linked to 'fast'- and 'slow'-equilibrating peripheral compartments. RESULTS: The model adequately described the changing plasma concentrations of endogenous L-carnitine in individual haemodialysis patients. Based on pooled data (mean +/- SD; n = 12), the volume of the central compartment was 10.09 +/- 0.72 l and the transfer rate constants into and out of the slowly equilibrating pool were 0.100 +/- 0.018 h(-1) and 0.00014 +/- 0.00016 h(-1), respectively. The turnover time of L-carnitine in the slow pool (which was assumed to represent muscle) was approximately 300 days. The model was in general agreement with separate data on the measured loss of carnitine from muscle in dialysis patients. CONCLUSIONS: Haemodialysis causes rapid reductions in plasma L-carnitine concentrations with each dialysis session. Plasma concentrations are restored between sessions by redistribution from peripheral compartments. However, during chronic haemodialysis, the ongoing dialytic loss of L-carnitine may lead to a slow depletion of the compound, contributing to a possible secondary deficiency.

Accumulation of trimethylamine and trimethylamine-N-oxide in end-stage renal disease patients undergoing haemodialysis.

BACKGROUND: Trimethylamine (TMA) is a short-chain tertiary aliphatic amine that is derived from the diet either directly from the consumption of foods high in TMA or by the intake of food high in precursors to TMA, such as trimethylamine-N-oxide (TMNO), choline and L-carnitine. The clinical significance of TMA may be related to its potential to contribute to neurological toxicity and 'uraemic breath' in patients with end-stage renal disease (ESRD). METHODS: Concentrations of TMA and TMNO in plasma from 10 healthy adults (not on haemodialysis) and 10 adults with ESRD undergoing haemodialysis (pre- and post-dialysis) were determined by gas chromatography-mass spectrometry. RESULTS: The concentrations of TMA and TMNO in pre-dialysis plasma (1.39+/-0.483 and 99.9+/-31.9 microM, respectively) were significantly (P<0.05) higher than the corresponding levels in healthy subjects (0.418+/-0.124 and 37.8+/-20.4 microM, respectively). However, there were no significant differences between post-dialysis and healthy subject plasma concentrations. In the ESRD patients, there was a significant (P<0.05) reduction in plasma TMA (from 1.39+/-0.483 to 0.484+/-0.164 microM) and TMNO (from 99.9+/-31.9 to 41.3+/-18.8 microM) during a single haemodialysis session. CONCLUSIONS: TMA and TMNO accumulate between haemodialysis sessions in ESRD patients, but are efficiently removed during a single haemodialysis session.

Impact of haemodialysis on individual endogenous plasma acylcarnitine concentrations in end-stage renal disease.

BACKGROUND: Patients with end-stage renal disease (ESRD) undergoing long-term haemodialysis exhibit low L-carnitine and elevated acylcarnitine concentrations. This study evaluated endogenous concentrations of an array of acylcarnitines (carbon chain length up to 18) in healthy individuals and ESRD patients receiving haemodialysis, and examined the impact of a single haemodialysis session on acylcarnitine concentrations. METHODS: Blood samples were collected from 60 healthy subjects and 50 ESRD patients undergoing haemodialysis (pre- and post-dialysis samples). Plasma samples were analysed for individual acylcarnitine concentrations by electrospray MS/MS. RESULTS: Of the 31 acylcarnitines, 29 were significantly (P<0.05) elevated in ESRD patients compared with healthy controls; in particular, C5 and C8:1 concentrations were substantially elevated. For acylcarnitines with a carbon chain length less than eight, plasma acylcarnitine concentrations decreased significantly over the course of a single dialysis session; however, post-dialysis concentrations invariably remained significantly higher than those in healthy subjects. Dialytic removal of acylcarnitines diminished once the acyl chain length exceeded eight carbons. CONCLUSIONS: The accumulation of acylcarnitines during long-term haemodialysis suggests that removal by haemodialysis is less efficient than removal from the body by the healthy kidney. Removal is significantly correlated to acyl chain length, most likely due to the increased molecular weight and lipophilicity that accompanies increased chain length.

Trimethylamine: metabolic, pharmacokinetic and safety aspects.

Trimethylamine (TMA) is a volatile tertiary aliphatic amine that is derived from the diet either directly from the consumption of foods containing TMA, or by the intake of food containing precursors to TMA such as trimethylamine-N-oxide (TMNO), choline and L-carnitine. Following oral absorption in humans, TMA undergoes efficient N-oxidation to TMNO, a reaction catalyzed by the flavin-containing monooxygenase (FMO) isoform 3 enzyme. TMNO subsequently undergoes excretion in the urine, although, evidence also suggests that metabolic retro-reduction of TMNO can occur. Whilst the pharmacokinetics of TMA and TMNO has not been fully elucidated in humans, a number of studies provide information on the likely fate of dietary derived TMA. Trimethylaminuria is a condition that is characterized by a deficiency in FMO3 enzyme activity, resulting in the excretion of increased amounts of TMA in bodily fluids such as urine and sweat, and breath. A human FMO3 database has been established and currently twenty-eight variants of the FMO3 gene have been reported including twenty-four missense, three nonsense, and one gross deletion mutation. Whilst TMA and TMNO are generally regarded as non-toxic substances, they are of clinical interest because of their potential to form the carcinogen N-nitrosodimethylamine.

Impact of hemodialysis on endogenous plasma and muscle carnitine levels in patients with end-stage renal disease.

BACKGROUND: End-stage renal disease (ESRD) patients undergoing hemodialysis treatment have reduced plasma L-carnitine levels; however, the relationship between dialysis age and carnitine status is poorly understood. This study examined the relationship between duration of dialysis and plasma and skeletal muscle concentrations of L-carnitine and its esters in ESRD patients. METHODS: Blood samples were collected from 21 patients at baseline and throughout the first 12 months of hemodialysis. In 5 patients, muscle samples were obtained after 0, 6, and 12 months of hemodialysis. Blood and muscle samples were collected from an additional 20 patients with a mean dialysis age of 5.10 years. L-carnitine, acetyl-L-carnitine, and total L-carnitine were measured by high-performance liquid chromatography (HPLC). RESULTS: The mean +/- SD plasma L-carnitine concentration in ESRD patients who had not yet started hemodialysis was 50.6 +/- 20.0 micromol/L. Significantly lower concentrations were observed after 12 months (29.7 +/- 10.5 micromol/L) and >12 months (22.0 +/- 5.4 micromol/L) of hemodialysis treatment. Acetyl-L-carnitine also declined with dialysis age, while plasma nonacetylated acylcarnitines continued to increase with the progression of hemodialysis therapy. An inverse relationship between dialysis age and muscle L-carnitine concentrations was observed. CONCLUSION: Long-term hemodialysis treatment is associated with a significant reduction in endogenous plasma and muscle L-carnitine levels and a significant increase in plasma acylcarnitines. The majority of the change in plasma L-carnitine concentrations occurs within the first few months of hemodialysis, while muscle levels continue to decline after 12 months of treatment.

Pharmacokinetics of L-carnitine.

L-Carnitine is a naturally occurring compound that facilitates the transport of fatty acids into mitochondria for beta-oxidation. Exogenous L-carnitine is used clinically for the treatment of carnitine deficiency disorders and a range of other conditions. In humans, the endogenous carnitine pool, which comprises free L-carnitine and a range of short-, medium- and long-chain esters, is maintained by absorption of L-carnitine from dietary sources, biosynthesis within the body and extensive renal tubular reabsorption from glomerular filtrate. In addition, carrier-mediated transport ensures high tissue-to-plasma concentration ratios in tissues that depend critically on fatty acid oxidation. The absorption of L-carnitine after oral administration occurs partly via carrier-mediated transport and partly by passive diffusion. After oral doses of 1-6g, the absolute bioavailability is 5-18%. In contrast, the bioavailability of dietary L-carnitine may be as high as 75%. Therefore, pharmacological or supplemental doses of L-carnitine are absorbed less efficiently than the relatively smaller amounts present within a normal diet.L-Carnitine and its short-chain esters do not bind to plasma proteins and, although blood cells contain L-carnitine, the rate of distribution between erythrocytes and plasma is extremely slow in whole blood. After intravenous administration, the initial distribution volume of L-carnitine is typically about 0.2-0.3 L/kg, which corresponds to extracellular fluid volume. There are at least three distinct pharmacokinetic compartments for L-carnitine, with the slowest equilibrating pool comprising skeletal and cardiac muscle.L-Carnitine is eliminated from the body mainly via urinary excretion. Under baseline conditions, the renal clearance of L-carnitine (1-3 mL/min) is substantially less than glomerular filtration rate (GFR), indicating extensive (98-99%) tubular reabsorption. The threshold concentration for tubular reabsorption (above which the fractional reabsorption begins to decline) is about 40-60 micromol/L, which is similar to the endogenous plasma L-carnitine level. Therefore, the renal clearance of L-carnitine increases after exogenous administration, approaching GFR after high intravenous doses. Patients with primary carnitine deficiency display alterations in the renal handling of L-carnitine and/or the transport of the compound into muscle tissue. Similarly, many forms of secondary carnitine deficiency, including some drug-induced disorders, arise from impaired renal tubular reabsorption. Patients with end-stage renal disease undergoing dialysis can develop a secondary carnitine deficiency due to the unrestricted loss of L-carnitine through the dialyser, and L-carnitine has been used for treatment of some patients during long-term haemodialysis. Recent studies have started to shed light on the pharmacokinetics of L-carnitine when used in haemodialysis patients.