Amino Keto Acids Research Articles

Abstract 433: BCAT1 promotes HCC metabolic reprogramming and survival through HIF-1α stabilization

Abstract Hepatocellular carcinoma (HCC) is a highly lethal primary liver malignancy characterized by hypoxia, a common feature of solid tumors, particularly in HCC. Hypoxia-inducible factors (HIFs) play a pivotal role in mediating various cellular processes, such as metabolic adaptation, to support the survival of hypoxic cells. The cytosolic form of branched-chain amino transferase (BCAT1) is responsible for the reversible transamination reaction between branched-chain keto-amino acids (BCKAs) and branched-chain amino acids (BCAAs), facilitating the interconversion between α-ketoglutarate (α-KG) and glutamate. Interestingly, we found that in the context of HCC, BCAT1 consumes α-KG, thereby favoring the stabilization of HIF-1α through suppressing the α-KG-dependent prolyl hydroxylase (PHD). Knockdown (KD) or knockout (KO) of BCAT or BCAT inhibitor (ERG245) suppressed HIF-1 stability, as well as HIF-1 dependent transcriptomic and metabolic changes. Mass spectrometry (MS)-based isotopic carbon tracing showed that KD/KO or ERG 245 suppressed hypoxia/HIF-1-induced glycolytic flux. Interestingly, ERG245 effectively inhibited the proliferative output of hypoxic HCC cells and suppressed HCC growth. ERG245 also profoundly inhibited HCC growth in vitro and in vivo together with Sorafenib, a tyrosine kinase inhibitor (TKI) used in HCC by increasing the apoptotic cell death. Furthermore, we confirmed that the expressions of BCAT1 and the HIF-1α-targeted metabolic genes are positively correlated in human HCCs. Our findings unequivocally demonstrate that BCAT1 confers a growth advantage to HCC cells by stabilizing HIF-1α and orchestrating HIF-1α-mediated metabolic reprogramming. Consequently, targeting BCAT1 emerges as a promising therapeutic strategy for HCC patients. Funding Support: This work was supported by the Centre for Oncology and Immunology under the Health@InnoHK initiative funded by the Innovation and Technology Commission, The Government of Hong Kong SAR, China and National Natural Science Foundation of China (NSFC) (82173123, C.C.-L.W.) Citation Format: Misty Shuo Zhang, Kenneth Kin-Leung Kwan, Weixing Li, Adonia E. Papathanassiu, Irene Oi-Lin Ng, Carmen Chak-Lui Wong. BCAT1 promotes HCC metabolic reprogramming and survival through HIF-1α stabilization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 433.

Alteration of Inflammation Marker Levels with Alfa Keto Analogs in Diabetic Rats

Diabetes mellitus is the most well-known and common cause of end-stage renal disease. Excessive inflammatory processes were hypothesized to be one of the reasons for progression to end-stage disease. Even though progression to end stage disease tried to be prevented with some dietary measures such as lowering nitrogen in diet, none of the methods tried were successful enough. In our study, we aimed to determine the effects of alfa keto analog use in altering levels of inflammatory markers when added to dietary program in a diabetic rat model. The study was performed on 22 male Sprague Dawley rats with streptozocine induced diabetic nephropathy. Both groups were fed with low protein diet except for study group with added alfa keto analogs. Biochemical values and inflammatory markers were studied with ELISA assay. Significant difference in serum albumin was found between study group and control group following administration of alfa keto analogs (p <.001). Also mentioned dietary modification made a significant difference in suppression of inflammatory reactions for interleukin (IL)-1, IL-6, IL-10, IL-18 and tumor necrosis factor-α. Adding keto amino acids to diets that are already low on protein, can slow progression to end-stage renal disease by reducing inflammation and protein loss in an animal model.

Phenylbutyrate, a branched-chain amino acid keto dehydrogenase activator, promotes branched-chain amino acid metabolism and induces muscle catabolism in C2C12 cells.

New Findings What is the central question of this study? The compound sodium phenylbutyrate (PB) has been shown to promote branched‐chain amino acid (BCAA) catabolism, and as such has been proposed as a treatment for disorders with enhanced BCAA levels: does PB induce muscle protein catabolism by forcing BCAA degradation away from muscle protein synthesis and mechanistic target of rapamycin (mTOR) inhibition? What is the main finding and its importance? Accelerated BCAA catabolism using PB resulted in adverse effects related to mTOR signalling and muscle protein metabolism in skeletal muscle cells, which may limit its application in conditions where muscle wasting is a risk. The compound sodium phenylbutyrate (PB) has been used for reducing ammonia in patients with urea cycle disorders and proposed as a treatment for disorders with enhanced branched‐chain amino acid (BCAA) levels, due to its effects on promoting BCAA catabolism. In skeletal muscle cells, we hypothesised that PB would induce muscle protein catabolism due to forcing BCAA degradation away from muscle protein synthesis and downregulating mechanistic target of rapamycin (mTOR). PB reduced medium BCAA and branched‐chain keto acid (BCKA) concentrations, while total cell protein (−21%; P < 0.001 vs. control) and muscle protein synthesis (−25%; P < 0.001 vs. control; assessed by measurement of puromycin incorporation into polypeptides) were decreased with PB. The regulator of anabolic pathways mTOR and its downstream components were impaired with PB treatment. The present results indicate that accelerated BCAA catabolism using PB resulted in adverse effects related to mTOR signalling and muscle protein metabolism, which may limit its application in settings where muscle wasting is a risk.

Exploratory-Data and Statistical Analyses of AB070597, an Amino Acid/Peptide Complex, on Blood-Serum Creatinine Concentration and Estimated Glomerular Filtration Rate: A Non-Randomized Pilot Trial of Five Humans with Declining Renal Function

ABSTRACT&#x0D; Background:&#x0D; Chronic kidney disease (CKD) develops from persistent, irremediable loss of renal function (RF). Animal studies show that dietary supplement, AB070597, an amino acid/ peptide complex, can stabilize, and in some instances, reverse CKD. Previous human CKD studies demonstrate that dietary treatment with specific keto acids and amino acids can lower blood-serum creatinine concentration (SCr) and increase glomerular filtration rate (GFR). This pilot trial was performed to determine whether a future randomized controlled human clinical trial of AB070597’s effect on CKD was justified.&#x0D; Methods: &#x0D; The trial was structured as a consecutive case series to evaluate whether oral treatment with AB070597 could slow CKD progression, as gaged by SCr, and estimated glomerular filtration rate (eGFR). Eligibility requirements: Non-diabetic white males under current medical care in the United States, diagnosed with CKD, or with histories of increasing SCr and declining eGFR, resulting from CKD or natural consequence of aging. Exclusion criteria: Concurrent or suspected comorbidities unrelated to CKD. Participants ingested 6-g bi-daily doses of AB070597 for periods up to 24 months. SCr was measured and eGFR calculated at approximate tri-monthly intervals. &#x0D; Results:&#x0D; Results were presented as individual-participant and group SCr and eGFR vectors, and initial-data-analysis diagrams.&#x0D; Treatment reversed SCr slopes from positive to negative, and eGFR slopes from negative to positive. When individual participant’s median monthly eGFR rate-of-change (positive) was compared with the median monthly eGFR rate-of-change (negative) in a sample of 2870 humans with CKD, differences were significant in favor of the AB070597 treatment group in 4 of 5 participants: (P = 0.0625, 95% CI: 0.368-8.368), (P = 0.0313, 95% CI: 0.368-1.038), (P = 0.0039, 95% CI: 0.368-0.698), (P = 0.0010, 95% CI: 0.118-2.868),&#x0D; (P &lt;0.0001, 95% CI: 0.368-10.370).&#x0D; Conclusion:&#x0D; Oral AB070597 treatment produced an apparently favorable change in CKD trajectory in 4 humans and in 1 with naturally age-related declining RF. The magnitude and direction of change hint that treatment may have had a beneficial effect on CKD progression and age-related RF. In and of themselves, this pilot trial’s results seem to favor CKD treatment with AB070597, but because there was no randomization, no control group, and a small sample size , it is not possible to extend results beyond its bounds. They do, however, support the rationale for a future randomized controlled trial

Myostatin in protein-energy wasting in patients on hemodialysis

The aim : to assess the effect of myostatin (MSTN) on the development of clinical and laboratory manifestations of protein metabolism disorders in patients receiving hemodialysis. Patients and methods : 80 patients with CKD 5D stage (47 males and 33 females) were treated with programmed hemodialysis. All patients underwent a thorough collection of clinical and anamnesis data, laboratory monitoring with the determination of the level of MSTN in the blood by enzyme immunoassay, dynamometric and bioelectrical impedance research. Statistical analysis was performed using “STATISTICA 10.0” (“StatSoft Inc.”, USA) and “Microsoft Office Excel 2010” (“Microsoft Corp.”, USA). Results : the regression analysis did not reveal a statistically significant association of MSTN levels with clinical indicators of protein-energy insufficiency (PED), albumin, β2-microglobulin, CRP, and blood ferritin. Neither the use of various drugs (with the exception of ketoamino acids), nor the presence of a concomitant pathology in history, did not change the level of MSTN. At the same time, the dependence of the local increase in the thickness of the skin folds with the test marker was revealed, the increase in which is associated with a progressive decrease in muscle strength. Conclusion : as a result of the study, the relationship between the serum concentration of myostatin and the clinical and laboratory manifestations of PEM was evaluated. The obtained data characterize the contribution of MSTN to the process of muscle protein degradation, which may become a target for targeted therapeutic effects and requires further study.

Stable Europium(III) Complexes with Short Linkers for Site-Specific Labeling of Biomolecules.

In this study, two new terpyridine‐based EuIII complexes were synthesized, the structures of which were optimized for luminescence resonance energy‐transfer (LRET) experiments. The complexes showed high quantum yields (32 %); a single long lifetime (1.25 ms), which was not influenced by coupling to protein; very high stability in the presence of chelators such as ethylenediamine‐N,N,N′,N′‐tetraacetate and ethylene glycol‐bis(2‐aminoethylether)‐N,N,N′,N′‐tetraacetic acid; and no interaction with cofactors such as adenosine triphosphate and guanosine triphosphate. A special feature is the short length of the linker between the EuIII ion and the maleimide or hydrazide function, which allows for site‐specific coupling of cysteine mutants or unnatural keto amino acids. As a consequence, the new complexes appear particularly suited for accurate distance measurements in biomolecules by LRET.

Optimisation of a system for the co-translational incorporation of a keto amino acid and its application to a tumour-specific Anticalin

The bioorthogonal keto group has attracted interest for the site-specific chemical conjugation of recombinant proteins under mild conditions, e.g. with aminooxy-functionalised fluorescent probes, radiometal chelates, toxins or polymers. However, the cotranslational incorporation of the corresponding non-canonical amino acid p-acetyl-L-phenylalanine (Apa) into proteins expressed in Escherichia coli by means of amber suppression using a previously described system with a mutated tRNA and an engineered tyrosyl-tRNA synthetase from Methanococcus jannaschii shows limited efficiency and considerable promiscuity towards endogenous amino acids. Employing a one-plasmid system that encodes all three components required for selection, i.e. the modified aminoacyl-tRNA synthetase (aaRS), the cognate amber suppressor tRNA and the enhanced green fluorescent protein equipped with an amber stop codon and serving as reporter, we have generated an Apa-specific aaRS&tRNA pair with considerably improved efficiency (17-fold increased expression) and also fidelity (6-fold). To this end, both the aaRS and the tRNA were subjected to doped random mutagenesis and selection in altogether four evolutionary cycles using fluorescence-activated bacterial cell sorting as well as automated screening of microcultures. The resulting aaRS&tRNA pair was applied to the functionalisation of an Anticalin with specificity towards oncofetal fibronectin by introducing a keto group at a permissible site for subsequent conjugation with a fluorescent dye, thus allowing visualisation of this tumour target under the microscope.

BENEFICIAL EFFECT OF KETO AMINO ACIDS FOR DIALYSIS PATIENTS

Nutritional status is an important predictor of clinical outcome in dialysed patients. Beside decreased serum protein/albumin,lower BMI with decreased muscle mass is the most significant predictor of morbidity and mortality. Keto amino acids (KA) represent an additional source for protein anabolism influencing indirectly also carbohydrate and lipid metabolism,Ca-P and acid base balance.Additionaly,by concominant metabolic and hemodynamic effect on residual nefrons, KA can help to slow progression of residual renal function (RRF) mainly in peritoneal dialysis patients. We conducted a long-term prospective randomized placebo controlled trial to test whether a modified low-protein diet (LPD) with or without keto acids (KA) would be safe ,well tolerated and associated with an increase of metabolic status and preservation of RRF in peritoneal dialysis (PD). We evaluated a total of 62 PD patients (32M/30F) aged 26-72 yrs with creatinine clearance (Ccr) 7.9-5.7 mL/min/1.73m2 for a period of 12 months. All patients were on modified LPD containing 0.8 protein/kg/IBW/day and 135/kJ/kg/IBW/day. LPD was randomly supplemented with KA at dosage of 100 mg/kg/IBW/day (30 patients, Group I) while 30 patients (Group II) received placebo. We analysed also muscle and fat metabolism by MR spectroscopy (MRS, m.tibialis anterior)) and imagining (MRI,visceral fat).Patients from Group I were before enrolment on conservative management using LPD + KA (0.6g P + 0.1g KA/kg/IBW/day) for longer time (18-48 months, median 28) with good compliance (SGA). Patients from group II were never treated with LPD and KA.All patients were monitored at the beginning of PD and at every 3 months for 12 months.;A neutral or positive long- term nitrogen balance (nPCR in g/kg IBW/day) was achieved in Group I (p

Effect of keto amino acids on asymmetric dimethyl arginine, muscle and fat tissue in chronic kidney disease

Levels of endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) are elevated in chronic kidney disease (CKD) and may contribute to vascular complications. In this study we tested the hypothesis that elevated ADMA can be reduced in CKD patients by long-term administration of low-protein diet (LPD) supplemented with keto amino acids (KA). In a long-term prospective double blind placebo controlled randomized trial, we evaluated a total of 120 CKD patients (62/58F) aged 22-76 yrs with creatinine clearance 22-40mL/min/1.73m 2 for a period of 36 months. All patients were on low-protein diet containing 0.6 protein/kg/IBW/day and 120-125/kJ/kg/IBW/day. LPD was randomly supplemented with KA at dosage of 100 mg/kg/IBW/day (61 patients, Group I) while 59 patients (Group II) received placebo. During the study period, glomerular filtration rate (GFR) slightly decreased (C cr from 34.2±11.6 to 29.9±9.2 mL/min and 33.5±11.6 to 22.2±10.4 mL/min in Group I and II, respectively); this however was more marked in Group II (p<0.01). Fat in muscle measured by MR spectroscopy (MRS, m.tibialis anterior) significantly decreased in Group I and was linked to reduced volume of visceral fat measured by MRI (p<0.01). Reduction of fat in Group II was not significant. In Group I, there was a significant decrease in the plasma level of ADMA (from 2.4±0.4 to 1.2±0.3 μmol/L, p<0.01), but ADMA remained unchanged in Group II. A further remarkable finding was reduction in the plasma concentration of pentosidine (from 486±168 to 325±127 μg/L, p<0.01) and decrease of proteinuria (from 3.7±2.20 to 1.6±1.2 g/24hrs, p<0.01) in Group I. Plasma adiponectin (ADPN) in Group I rose (p<0.01). Analysis of lipid spectrum revealed a mild yet significant decrease in total cholesterol and LPD-cholesterol (p<0.01), more pronounced in Group I. In Group I, there was a decrease in plasma triglycerides (from 3.8±1.5 down to 2.3±0.5 mmol/L, p<0.01), whereas glycated hemoglobin (HbAc1) decreased from 7.0±1.3 to 4.1±0.9 %, (p<0.01) nIn conclusion,comparing to placebo group long term co-administration of LPD and KA in CKD patients led to decrease of ADMA,fat in muscle and visceral body fat, and proteinuria. Concomitant decreases of glycated haemoglobin, LDL-c and pentosidine may also contribute to the delay in progression of renal failure and decrease of cardiovascular risk factors..The study was supported by Research Project MZO 00023001 awarded by Ministry of Health of the Czech Republic

ANTI-INFLAMMATORY EFFECTS OF LOW PROTEIN DIET SUPPLEMENTED WITH KETO-AMINO ACID IN THE TREATMENT OF TYPE 2 DIABETIC NEPHROPATHY

Recent clinical research strongly approves that low-protein diet supplemented with keto-amino acid can effectively delay progression of type 2 diabetic nephropathy (DN). Anti-inflammation is one of these effects, but the mechanism is still controversial. This study is designed to further explore roles of ketogenic diets in regulation of inflammation status of type 2 DN. Twenty-one patients with type 2 DN (mean age at 65.14±7.34 years), were followed-up for 52 weeks in this study. All patients were in CKD stages 3–4 with glomerular filtration rates 26–55 ml/min/1.73 m2 and were all on a low-protein diet containing 0.8 g protein/kg BW per day and 30–35 Kcal /kg BW per day. The diet was randomly supplemented with keto-amino acids at a dosage of 100 mg/kg BW per day in 10 patients, who were assigned into Group II. Other 11 patients were assigned into Group I. At the end of this study, related clinical data showed there was a significant increase in the serum level of TNF- α which could mediate inflammation systemically in Group I (from 230.25±54.34 to 332.11 pg/ml, P < 0.01), but non-significant increase in Group II (from 224.59±41.24 to 253.41±31.28 pg/ml, P>0.05). The level of CRP, which is produced in response to inflammation, rose greatly in Group I (from 7.5±1.07 to 20.4±3.72 ug/ml, P < 0.01), but decreased in Group II (from 8.2±3.07 to 3.9±1.22 ug/ml, P < 0.01). The level of adiponectin, an anti-inflammatory factor, was decreased in Group I (from 9.42±0.8 to 7.64±1.4 μg/ml, P< 0.05), but showed slight increase in Group II (9.04±0.9 versus 10.47±1.2= μg/ml, P>0.05). Nutritional markers including serum albumin, hemoglobin and basal metabolic index showed no malnutrition happened during the follow-up period. In conclusion, low-protein diet supplemented with keto-amino acids contribute to ameliorate inflammation in the progression of type 2 diabetic nephropathy through regulating inflammatory factors production, including TNF- α , CRP and adiponectin.

EFFECTS OF A KETO/AMINO ACID SUPPLEMENTED LOW PROTEIN DIET ON THE DELAY OF PROGRESSIEVE RENAL FAILURE IN CHRONIC KIDNEY DISEASE

A protein-restricted diet with keto/amino acids (KA) supplement showed favorable effects on delayed renal replacement therapy in patients with chronic kidney disease. This is an open, prospective, randomized, and multi-center study. A total of 67 patients were randomly assigned into two groups. LPD+KA group was advised to take less than 0.6 g/kg/day of protein with KAs. LPD group was advised to consume less than 0.6 g/kg/day protein. Nutritional and clinical parameters were evaluated at baseline, 3 and 6 months. Nutritional status represented as body mass index, mid-arm circumference and triceps skin-fold thickness was not different between the two groups at 3months and 6 months. Ca×P product level measured at 3 months was lower in the LPD+KA group than in the LPD group (LPD+KA group: 33.5±5.0 vs. LPD group: 36.9±7.9 mg 2 /dL 2 , p

STUDY MECHANISMS OF LOW PROTEIN DIET SUPPLEMENTED WITH KETOANLOGS ON REDUCING PROTEINURIA AND MINTINING NUTRITIONAL STATUS IN TYPE2 DIBETIC NEPHROPTHY

Diabetic nephropathy is one of the most important causes of end stage renal disease (ESRD) in the world. Recently in a multicenter, randomized clinical trial performed in China, it was found that compound α-keto acid tablet in combination with low protein diet(LPD+KA) reduced proteinuria and delayed the progression to ESRD in Type 2 diabetic nephropthy(T2DN). However, the mechanisms underlined these effects were not been elucidated. This study is a part of the continuation of the study to explore the effects of LPD+KA on podocyte as well as on local RAS in the kidney. A total of 60 patients with T2DN and CKD stages 3–4 are included in this study. All patients are treated with a LPD containing 0.6g protein/kg BW per day and 120–125 kJ/kg BW per day. The diet is randomly supplemented with keto-amino acids (Ketosteril®, Fresenius Kabi) at a dosage of 100 mg/kg BW per day. The other 30 patients receive placebo. After six months of follow-up, protein intake differed only by 0.07 g/kg/day between the two groups. The mean declines in GFR were 4.2 mL/min/year (95%CI;2.9 to 5.5) in LPD group and 3.7 (95%CI; 2.7 to 4.7) in LPD+KA group. Compared with LPD, paitients in LPD+KA group had reduced proteinuria (2.16±0.4 vs 3.56±0.6 g/day; p〈0.05). However, serum albumin and prealbumin levels did not change. Through further research we found that, after a median follow-up for six months, the urinary angiotensinogen:creatinine ratio in LPD patients was higher than in LPD+KA subjects (10.1 ±3.2 μg/g vs 5.8 ± 1.2 μg/g; p〈0.05);The urinary mRNA levels of the target genes nephrin, podocin and synaptopodin of podocyte were higher in the LPD group compared with LPD+KA(p〈0.05) in urinary sediment, and the expression of nephrin, podocin and synaptopodin mRNA positively correlated with serum creatinine (r=0.326, p = 0.04; r = 0.426, p = 0.03; r = 0.343, p = 0.001, respectively). In conclusion, compound α-keto acid tablet in combination with low protein diet (LPD+KA) is associated with amelioration of proteinuria, better reduction in the loss of kidney function compared with LPD alone meanwhile nutrition status remained well. The mechanism under these effects may be explained by the role of LPD+KA diet in reducing urine podocyte loss and lowering the angiotensinogen level in the urine.

Expanding the Genetic Repertoire of the Methylotrophic Yeast Pichia pastoris

To increase the utility of protein mutagenesis with unnatural amino acids, a recombinant expression system in the methylotrophic yeast Pichia pastoris was developed. Aminoacyl-tRNA synthetase/suppressor tRNA (aaRS/tRNA(CUA)) pairs previously evolved in Saccharomyces cerevisiae to be specific for unnatural amino acids were inserted between eukaryotic transcriptional control elements and stably incorporated into the P. pastoris genome. Both the Escherichia coli tyrosyl- and leucyl-RS/tRNA(CUA) pairs were shown to be orthogonal in P. pastoris and used to incorporate eight unnatural amino acids in response to an amber codon with high yields and fidelities. In one example, we show that a recombinant human serum albumin mutant containing a keto amino acid (p-acetylphenylalanine) can be efficiently expressed in this system and selectively conjugated via oxime ligation to a therapeutic peptide mimetic containing an permittivity-(2-(aminooxy)acetyl)-L-lysine residue. Moreover, unnatural amino acid expression in the methylotrophic host was systematically optimized by modulation of aaRS levels to express mutant human serum albumin in excess of 150 mg/L in shake flasks, more than an order of magnitude better than that reported in S. cerevisiae. This methodology should allow the production of high yields of complex proteins containing unnatural amino acids whose expression is not practical in existing systems.

Reduction of plasma asymmetric dimethylarginine in obese patients with chronic kidney disease after three years of a low-protein diet supplemented with keto-amino acids: a randomized controlled trial

Levels of the endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) are elevated in chronic kidney disease (CKD) and may contribute to vascular complications. In this study we tested the hypothesis that elevated ADMA can be reduced in obese CKD patients by long-term administration of a low-protein diet supplemented with keto-amino acids. In a long-term prospective double-blind placebo-controlled randomized trial, we evaluated for a period of 36 months a total of 111 CKD patients (54 men, 57 women) aged 22-76 years with obesity (BMI >or= 30 kg/m(2)) and an inulin clearance rate (C(in)) of 22-40 ml/min/1.73 m(2). All patients were on a low-protein diet containing 0.6 g protein/kg BW per day and 120-125 kJ/kg BW per day. The diet was randomly supplemented with keto-amino acids at a dosage of 100 mg/kg BW per day (66 patients, Group I); 65 patients received placebo (Group II). During the study period, the glomerular filtration rate decreased slightly in Group I (C(in) from 32.4 +/- 12.6 to 29.8 +/- 8.6 ml/min/1.73 m(2)) and more markedly in Group II (from 33.2 +/- 12.6 to 23.2 +/- 98.4 ml/min/1.73 m(2), P < 0.01). BMI decreased significantly in Group I (from 32.0 +/- 3.3 to 26.1 +/- 4.0 kg/m(2), P < 0.01) and was linked to reduced volume of visceral fat measured by MRI (P < 0.01). Reduction of BMI in Group II was not significant. In Group I, there was a significant decrease in the plasma level of ADMA (from 2.5 +/- 0.5 to 1.3 +/- 0.4 micromol/l, P < 0.01), but ADMA remained unchanged in Group II. A further remarkable finding in Group I was reduction in the plasma concentration of pentosidine (from 480 +/- 170 to 320 +/- 120 microg/l, P < 0.01) and decrease of proteinuria (from 3.8 +/- 2.24 to 1.6 +/- 1.0 g/24 h, P < 0.02). Plasma adiponectin rose in Group I (P < 0.01). Analysis of the lipid spectrum revealed a mild but significant decrease in total cholesterol and LPD-cholesterol (P < 0.02), more pronounced in Group I. There was also a decrease in plasma triglycerides in Group I (from 3.9 +/- 1.6 down to 2.2 +/- 0.6 mmol/l, P < 0.01) and a decrease in glycated hemoglobin (from 7.2 +/- 1.4% to 4.2 +/- 0.8%, P < 0.02). Compared with the placebo group, long term co-administration of a low-protein diet and keto-amino acids in CKD patients with obesity led to decreases of ADMA, visceral body fat and proteinuria. Concomitant decreases of glycated hemoglobin, LDL-cholesterol and pentosidine may also contribute to the delay in progression of renal failure.

Site-specific Incorporation of Keto Amino Acids into Functional G Protein-coupled Receptors Using Unnatural Amino Acid Mutagenesis

G protein-coupled receptors (GPCRs) are ubiquitous heptahelical transmembrane proteins involved in a wide variety of signaling pathways. The work described here on application of unnatural amino acid mutagenesis to two GPCRs, the chemokine receptor CCR5 (a major co-receptor for the human immunodeficiency virus) and rhodopsin (the visual photoreceptor), adds a new dimension to studies of GPCRs. We incorporated the unnatural amino acids p-acetyl-L-phenylalanine (Acp) and p-benzoyl-L-phenylalanine (Bzp) into CCR5 at high efficiency in mammalian cells to produce functional receptors harboring reactive keto groups at three specific positions. We obtained functional mutant CCR5, at levels up to approximately 50% of wild type as judged by immunoblotting, cell surface expression, and ligand-dependent calcium flux. Rhodopsin containing Acp at three different sites was also purified in high yield (0.5-2 microg/10(7) cells) and reacted with fluorescein hydrazide in vitro to produce fluorescently labeled rhodopsin. The incorporation of reactive keto groups such as Acp or Bzp into GPCRs allows their reaction with different reagents to introduce a variety of spectroscopic and other probes. Bzp also provides the possibility of photo-cross-linking to identify precise sites of protein-protein interactions, including GPCR binding to G proteins and arrestins, and for understanding the molecular basis of ligand recognition by chemokine receptors.

Efficient Syntheses of Biologically Important (S)-2-Amino-8-oxodecanoic Acid (Aoda) and Homologues

The efficient and practical asymmetric syntheses of the biologically important (S)-2-amino-8-oxodecanoic ester and its homologues have been achieved employing the Schöllkopf chiral auxiliary. Carbon-carbon bond formation between the appropriate alkyl bromide and the LDA generated anion of the Schöllkopf auxiliary, followed by hydrolysis provided the desired methyl ester of a long-chained keto amino acid in high yield with high selectivity.

Addition of the keto functional group to the genetic code of Escherichia coli.

Although the keto group is the most versatile of the functional groups in organic chemistry, it is absent in the genetically encoded amino acids. To overcome this natural limitation on protein biosynthesis, we have evolved an orthogonal tRNA-synthetase pair that makes possible the efficient incorporation of a keto amino acid, p-acetyl-l-phenylalanine, into proteins in E. coli with high translational fidelity in response to the amber nonsense codon. To demonstrate the utility of this keto amino acid, we have used it to modify a protein selectively with a small molecule fluorophore and biotin derivative. This additional genetically encoded amino acid should greatly expand our ability to manipulate protein structure and function both in vitro and in living cells.

Synthesis of an oxime-linked neoglycopeptide with glycosylation-dependent activity similar to its native counterpart

Neoglycopeptides containing an oxime sugar-peptide linkage can be generated by coupling an aminooxy sugar with a peptide bearing a keto-amino acid. The coupling reaction can be executed in aqueous milieu without need for protecting groups on the peptide or saccharide, or auxiliary coupling reagents. Using this method, an oxime-linked analog of an antimicrobial peptide with glycosylation-dependent function was prepared and found to have similar bioactivity to the native glycopeptide. Thus, replacement of the sugar-peptide bond with an unnatural but synthetically facile linkage can produce functional neoglycopeptides.

Amino acids with aryl‐keto function in their side chains

Amino acids with aryl-keto function in their side-chains were obtained in excellent yields in the reaction of omega-carboxyamino acids with liquid HF in the presence of aromatic compounds susceptible to electrophilic substitution, such as anisole, 2-methoxybiphenyl, butyl phenyl ether or 1,3-dimethoxybenzene. The new amino acids were converted smoothly into N-tert-butyloxycarbonyl or N-fluorenylmethoxycarbonyl derivatives and then incorporated into peptides by conventional coupling methods. During the coupling step, no formation of cyclic Schiff bases was observed for aryl-keto amino acids derived from DL-alpha-aminopimelic acid and from L-alpha-aminosuberic acid. In the crude products, truncated peptides terminated at the keto amino acids were not detected by liquid chromatography-mass spectrometry.

Serum phosphate control; what about calcium salts of keto-amino acids?

Journal Article Serum phosphate control; what about calcium salts of keto-amino acids? Get access M. Macia, M. Macia Nephrology DepartmentsH.N.S. de la Candelaria S/C de Tenerife, and H.U. San Carlos, Madrid, Spain Search for other works by this author on: Oxford Academic PubMed Google Scholar F. Coronel F. Coronel Nephrology DepartmentsH.N.S. de la Candelaria S/C de Tenerife, and H.U. San Carlos, Madrid, Spain Search for other works by this author on: Oxford Academic PubMed Google Scholar Nephrology Dialysis Transplantation, Volume 10, Issue 11, November 1995, Pages 2159–2160, https://doi.org/10.1093/ndt/10.11.2159 Published: 01 November 1995