Excretion Of Lysine Research Articles

Measuring bioavailability, utilization, and excretion of rumen-protected lysine in lactating cows using an isotope technique

Supplementing a diet with rumen-protected amino acids (AAs) is a common feeding strategy for efficient production. For a cost-effective use of rumen-protected AA, the accurate bioavailability of rumen-protected amino acids should be known and their metabolism after absorption needs to be well understood. The current study determined the bioavailability, absorption, utilization, and excretion of rumen-protected Lys (RP-Lys). Four ruminally cannulated cows in a 4 × 4 Latin square design (12 d for diet adaptation; 5 or 6 d for total collections) received the following treatments: L0, a basal diet; L25, the basal diet and L-Lys infused into the abomasum to provide 25.9 g/d L-Lys; L50, the basal diet and L-Lys infused into the abomasum to provide 51.8 g/d L-Lys; and RPL, the basal diet supplemented with 105 g/d (as-is) of RP-Lys to provide 26.7 g of digestible Lys. During the last 5 or 6 d in each period, 15N-Lys (0.38 g/d) was infused into the abomasum for all cows to label the pool of AA, and the total collection of milk, urine, and feces were conducted. 15N enrichment of samples on d 4 and 5 were used to calculate the bioavailability and Lys metabolism. We used a model containing a fast AA turnover (≤ 5 d) and slow AA turnover pool (> 5 d) to calculate fluxes of Lys. The Lys flux to the fast AA turnover pool (absorbed Lys + Lys from the slow AA turnover pool to fast AA turnover pool) was calculated using 15N enrichment of milk Lys. The flux of Lys from the fast AA turnover pool to milk and urine was calculated using 15N transfer into milk and urine. Then, absorbed Lys was estimated by the sum of Lys flux to milk and urine assuming no net utilization of Lys by body tissues. Duodenal Lys flow was estimated by 15N enrichment of fecal Lys. The bioavailability of RP-Lys was calculated from duodenal Lys flows and Lys absorption for RPL. Increasing Lys supply from L25 to L50 increased Lys utilization for milk by 9 g/d but also increased urinary excretion by 10 g/d. For RPL, absorbed Lys was estimated to be 136 g/d where 28 g of absorbed Lys originated from RP-Lys. In conclusion, 68% of bioavailability was obtained for RP-Lys. The Lys provided from RP-Lys was not only utilized for milk protein (48%) but also excreted in urine (20%) after oxidation.

Chitosan Increases Lysine Content through Amino Acid Transporters in Flammulina filiformis.

HighlightsChitosan treatment increased the lysine content of F. filiformis. Results from this study provide insights for industrial-scale trials potentially resulting in higher-quality F. filiformis.Response surface methodology (RSM) was used to optimize the conditions of chitosan-induced amino acid content in F. filiformis for the first time.The amino acid transporters in F. filiformis were systematically screened for the first time. The involvement of AAT3 and AAT4 genes in chitosan-induced lysine biosynthesis in F. filiformis was investigated by gene-silenced strains.Lysine content is considered an important indicator of the quality of Flammulina filiformis. In this study, chitosan was used to improve lysine content of F. filiformis. Optimal design conditions were obtained using central combination design (CCD): treatment concentration was 14.61 μg/mL, treatment time was 52.90 h, and the theoretical value of lysine content was 30.95 mg/g. We used Basic Local Alignment Search Tool Protein (BLASTP) to search the F. filiformis genome database using known AATs in the NCBI database. There were 11 members of AAT in F. filiformis. The expression levels of AAT3 and AAT4 genes increased significantly with chitosan treatment. Subsequently, AAT3 and AAT4 silencing strains were constructed using RNAi technology. The lysine content of the wild-type (WT) strain treated with chitosan increased by 26.41%. Compared with the chitosan-induced WT strain, chitosan-induced lysine content decreased by approximately 24.87% in the AAT3 silencing strain, and chitosan-induced lysine content in the AAT4 silencing strain increased by approximately 13.55%. The results indicate that AAT3 and AAT4 are involved in the regulation of the biosynthesis of lysine induced by chitosan in F. filiformis. AAT3 may participate in the absorption of lysine, and AAT4 may be involved in the excretion of lysine with chitosan treatment.

REFERENCE VALUES OF THE AMINO ACIDS CYSTINE, LYSINE AND ARGININE EXCRETION IN HEALTHY CHILDREN AND IN PATIENTS WITH DYSMETHABOLIC NEPHROPATHY

INTRODUCTION. For early diagnostics of metabolic disorders, which leads to dysmetabolic nephropathy (DMN) formation it is necessary to estimate excretion with urine lithogenic amino acids: a cystine, a lysine and arginine. THE AIM: to define reference excretion levels of cystine, arginine and lysine in daily and morning portions of urine at healthy children and to estimate their changes in patients with DMN. PATIENTS AND METHODS. 69 patients with DMN of 11-17 years and 600 healthy children participated in the research. General blood and urine tests, the biochemical analysis of urine with estimation of cystine, arginine and lysine were performed. The statistical analysis was carried out in the Statistica program (version 6.0). RESULTS. The excretion of cystine, lysine and arginine in daily urine increases with age. In a morning portion of urine – on the contrary: the highest levels in children under 1 year (cystine in boys 11,21±1,44, in girls – 11,28±1,60), and its decrease at the age of 15-17 years by 1,5-2,5 times both in boys (4,24±0,58), and in girls (7,89±0,63). In children with a dysmetabolic nephropathy all indices are significantly higher. CONCLUSION. Mean values of an excretion of cystine, arginine and lysine in daily urine increase with age and do not depend on sex. Excretion with urine all analyzed amino in patients with DMN was significantly higher than in healthy children. During clinical examination of children hereditary tainted by urolithiasis it is necessary to estimate excretion of the lithogenic amino acids in urine for identification of risk groups and early diagnostics of the metabolic disorders leading to dysmetabolic nephropathy and urolithiasis.

Impact of maternal obesity on the metabolic profiles of pregnant women and their offspring at birth

Obesity is currently an increasing public health problem. The intra-uterine environment plays a critical role in foetal development. The objective of this study is to investigate the association of obesity with modifications in the metabolic profiles of pregnant women, and their new-borns. Based on the PELAGIE cohort (Brittany, France), a sample of 321 pregnant women was divided into three groups according to their body mass index (BMI) (normal, over-weight and obese). Nuclear magnetic resonance-based metabolomics analyses were performed on maternal urine and cord-blood samples. Partial least squares regression-discriminant analysis (PLS-DA), polytomous and logistic regressions were used to differentiate the metabolic profiles of the three BMI groups after adjusting for potential confounders. Specific profiles were observed for the overweight and obese women (BMI > 25) compared to the normal-weight women: they had a decrease in urinary hippurate excretion associated with a decrease in phenylalanine and an increase in creatinine. We also showed an increase in the urinary excretion of lactate, citrate, acetate, creatine, and lysine only in obese women (BMI > 30) compared to the normal-weight women. The PLS-DA modelling did not reveal any significant difference between the cord-blood metabolic profiles of newborns according to maternal BMI—although infants born of obese women had a higher birth weight and a lower Apgar score. Our results confirmed the potential link between obesity and gut microbiota disruption (changes in urinary acids), as well as energy and amino-acid metabolism but did not reveal any disruption among newborns.

Systems metabolic engineering of Corynebacterium glutamicum for production of the chemical chaperone ectoine

BackgroundThe stabilizing and function-preserving effects of ectoines have attracted considerable biotechnological interest up to industrial scale processes for their production. These rely on the release of ectoines from high-salinity-cultivated microbial producer cells upon an osmotic down-shock in rather complex processor configurations. There is growing interest in uncoupling the production of ectoines from the typical conditions required for their synthesis, and instead design strains that naturally release ectoines into the medium without the need for osmotic changes, since the use of high-salinity media in the fermentation process imposes notable constraints on the costs, design, and durability of fermenter systems.ResultsHere, we used a Corynebacterium glutamicum strain as a cellular chassis to establish a microbial cell factory for the biotechnological production of ectoines. The implementation of a mutant aspartokinase enzyme ensured efficient supply of L-aspartate-beta-semialdehyde, the precursor for ectoine biosynthesis. We further engineered the genome of the basic C. glutamicum strain by integrating a codon-optimized synthetic ectABCD gene cluster under expressional control of the strong and constitutive C. glutamicum tuf promoter. The resulting recombinant strain produced ectoine and excreted it into the medium; however, lysine was still found as a by-product. Subsequent inactivation of the L-lysine exporter prevented the undesired excretion of lysine while ectoine was still exported. Using the streamlined cell factory, a fed-batch process was established that allowed the production of ectoine with an overall productivity of 6.7 g L-1 day-1 under growth conditions that did not rely on the use of high-salinity media.ConclusionsThe present study describes the construction of a stable microbial cell factory for recombinant production of ectoine. We successfully applied metabolic engineering strategies to optimize its synthetic production in the industrial workhorse C. glutamicum and thereby paved the way for further improvements in ectoine yield and biotechnological process optimization.

Differential cystine and dibasic amino acid handling after loss of function of the amino acid transporter b0,+AT (Slc7a9) in mice

Cystinuria is an autosomal recessive disease caused by mutations in SLC3A1 (rBAT) and SLC7A9 (b(0,+)AT). Gene targeting of the catalytic subunit (Slc7a9) in mice leads to excessive excretion of cystine, lysine, arginine, and ornithine. Here, we studied this non-type I cystinuria mouse model using gene expression analysis, Western blotting, clearance, and brush-border membrane vesicle (BBMV) uptake experiments to further characterize the renal and intestinal consequences of losing Slc7a9 function. The electrogenic and BBMV flux studies in the intestine suggested that arginine and ornithine are transported via other routes apart from system b(0,+). No remarkable gene expression changes were observed in other amino acid transporters and the peptide transporters in the intestine and kidney. Furthermore, the glomerular filtration rate (GFR) was reduced by 30% in knockout animals compared with wild-type animals. The fractional excretion of arginine was increased as expected (∼100%), but fractional excretions of lysine (∼35%), ornithine (∼16%), and cystine (∼11%) were less affected. Loss of function of b(0,+)AT reduced transport of cystine and arginine in renal BBMVs and completely abolished the exchanger activity of dibasic amino acids with neutral amino acids. In conclusion, loss of Slc7a9 function decreases the GFR and increases the excretion of several amino acids to a lesser extent than expected with no clear regulation at the mRNA and protein level of alternative transporters and no increased renal epithelial uptake. These observations indicate that transporters located in distal segments of the kidney and/or metabolic pathways may partially compensate for Slc7a9 loss of function.

Hyperexcretion of homocitrulline in a Malaysian patient with lysinuric protein intolerance

Lysinuric protein intolerance (LPI; MIM 222700) is an inherited aminoaciduria with an autosomal recessive mode of inheritance. Biochemically, affected patients present with increased excretion of the cationic amino acids: lysine, arginine, and ornithine. We report the first case of LPI diagnosed in Malaysia presented with excessive excretion of homocitrulline. The patient was a 4-year-old male who presented with delayed milestones, recurrent diarrhea, and severe failure to thrive. He developed hyperammonemic coma following a forced protein-rich diet. Plasma amino acid analysis showed increased glutamine, alanine, and citrulline but decreased lysine, arginine and ornithine. Urine amino acids showed a marked excretion of lysine and ornithine together with a large peak of unknown metabolite which was subsequently identified as homocitrulline by tandem mass spectrometry. Molecular analysis confirmed a previously unreported homozygous mutation at exon 1 (235 G > A, p.Gly79Arg) in the SLC7A7 gene. This report demonstrates a novel mutation in the SLC7A7 gene in this rare inborn error of diamino acid metabolism. It also highlights the importance of early and efficient treatment of infections and dehydration in these patients. The diagnosis of LPI is usually not suspected by clinical findings alone, and specific laboratory investigations and molecular analysis are important to get a definitive diagnosis.

Lysine triggers apoptosis through a NADPH oxidase‐dependent mechanism in human renal tubular cells

Progressive chronic kidney disease (CKD) is common in lysinuric protein intolerance (LPI), a primary inherited aminoaciduria characterized by massive Lysine excretion in urine. However, by which mechanisms Lysine may cause kidney damage to tubule cells is still not understood. This study determined whether Lysine overloading of human proximal tubular cells (HK-2) in culture enhances apoptotic cell loss and its associated mechanisms. Overloading HK-2 with Lysine levels reproducing those observed in urine of patients affected by LPI (10 mM) increased apoptosis (+30%; p < 0.01 vs.C), as well as Bax and Apaf-1 expressions (+30-50% p < 0.05), while downregulated Bcl-2 (-40% p < 0.05). Apoptosis induced by high Lysine was no longer observed after addition of caspase-9 and caspase-3 inhibitors while caspase-8 inhibitor had no protective effect. High Lysine induced elevations in ROS generation and NADPH oxidase subunits mRNAs (p22 (phox) +106 ± 23%, p67 (phox) +108 ± 22% and gp91 (phox) +75 ± 4% p < 0.05-0.01). In addition, the NADPH oxidase inhibitor DPI prevented both ROS production and apoptosis. Treating HK-2 with antioxidants, such as Cysteine and its analog, N-acetyl-L-cysteine (NAC), rescued the HK-2 from apoptosis induced by Lysine. In summary, our data show that high Lysine in vitro increases the permissiveness of proximal tubule kidney cells to apoptosis by triggering a pathway involving NADPH oxidase signaling. This event may represent a key cellular effect in the increasing the susceptibility of human tubular cells to apoptosis when the tubules cope with a high Lysine load. This effect is instrumental to renal damage and disease progression in patients with LPI.

Lysinuric protein intolerance: Reviewing concepts on a multisystem disease

Lysinuric protein intolerance (LPI) is an inherited aminoaciduria caused by defective cationic amino acid transport at the basolateral membrane of epithelial cells in intestine and kidney. LPI is caused by mutations in the SLC7A7 gene, which encodes the y(+)LAT-1 protein, the catalytic light chain subunit of a complex belonging to the heterodimeric amino acid transporter family. LPI was initially described in Finland, but has worldwide distribution. Typically, symptoms begin after weaning with refusal of feeding, vomiting, and consequent failure to thrive. Hepatosplenomegaly, hematological anomalies, neurological involvement, including hyperammonemic coma are recurrent clinical features. Two major complications, pulmonary alveolar proteinosis and renal disease are increasingly observed in LPI patients. There is extreme variability in the clinical presentation even within individual families, frequently leading to misdiagnosis or delayed diagnosis. This condition is diagnosed by urine amino acids, showing markedly elevated excretion of lysine and other dibasic amino acids despite low plasma levels of lysine, ornithine, and arginine. The biochemical diagnosis can be uncertain, requiring confirmation by DNA testing. So far, approximately 50 different mutations have been identified in the SLC7A7 gene in a group of 142 patients from 110 independent families. No genotype-phenotype correlation could be established. Therapy requires a low protein diet, low-dose citrulline supplementation, nitrogen-scavenging compounds to prevent hyperammonemia, lysine, and carnitine supplements. Supportive therapy is available for most complications with bronchoalveolar lavage being necessary for alveolar proteinosis.

Reduction of plasma taurine level in children affected by Osteogenesis Imperfecta during bisphosphonate therapy

Osteogenesis Imperfecta (OI) is a heritable disease of connective tissue characterized by increased bone fragility. To date, bisphosphonates seem to be the most promising therapy, at least for children. In the last decade experimental and clinical studies indicate that several amino acids are implicated in bone mineralization. Particularly, taurine is localized in matrices of the bone and can regulate osteoblast metabolism with antiosteopenic effect. To investigate a possible interaction between pharmacological effects of bisphosphonates and amino acids involved in bone metabolism, we performed plasma and urine amino acids analysis in children affected by OI before and during treatment with bisphosphonates. Fourteen prepubertal children with moderate to severe types of OI, 8 males and 6 females, aged from 2 to 11 years (mean (SD) 6,9 ± 2,53) were enrolled in the study. Patients were treated with neridronate infusion (1 mg/Kg/body weight) every three months. Plasma and urine specimens for amino acid analysis were kept at baseline (T0) and three months after each infusion of four consecutive cycles (T1–T4). A significant decrease in respect to the pre-treatment levels (T0) was observed after the fourth infusion for taurine ( p < 0.01). In addition, urinary excretion of this amino acid showed a significant decrease after the fourth infusion. No significant correlations were found between plasma level or urinary excretion of hydroxyproline, taurine, arginine and lysine in respect to bone mineral density. The progressive reduction of plasma taurine found in our patients treated with bisphosphonates could be implicated in the action mechanism of this drug in OI and possibly in other disorders of bone metabolism. This knowledge could provide new opportunities to improve treatment with bisphosphonates and address novel strategies for the therapeutic approach to bone disorders.

Effects of fat coated rumen bypass lysine and methionine on performance of dairy cows fed a diet deficient in lysine and methionine

ABSTRACTTwo experiments were conducted to evaluate the effect of fat coated rumen bypass lysine (RPLys) and methionine (RPMet) on the lactation performance of dairy cows. In Experiment 1, three lactating cows were supplied with RPLys and fat coated DL‐Met which was highly protected (H‐RPMet) as an indigestible marker, and total fecal emission was collected for 72 h following administration. Measuring the proportional difference in fecal excretion of lysine derived from RPLys relative to methionine derived from H‐RPMet, the intestinal availability of RPLys was estimated to be 66.2%. In Experiment 2, 20 multiparous Holstein cows producing approximately 40 kg/day of milk were assigned to two treatments; fed RPLys (16 g/day as lysine) and RPMet (6.5 g/day as methionine) or none (control) from 5 to 21 weeks postpartum. The consumption of dry matter, organic matter, crude protein, neutral detergent fiber and acid detergent fiber were significantly more in the control cows throughout the experimental period. Their milk protein yield, the contents of their milk protein and milk fat were higher by 0.03 kg (P = 0.03), 0.06% (P &lt; 0.001) and 0.11% (P = 0.07), respectively, in the treatment group compared to the control. These results suggest that the RPLys and RPMet used in this study improved the lactation performance of dairy cows.

The effect of tannic acid on the excretion of endogenous methionine, histidine and lysine with broilers

Tannins are a complex mixture of higher plant, water-soluble polyphenolic compounds of varying molecular masses that have the ability to react with proteins, polysaccharides and other macromolecules. Dietary tannins are thought to reduce the digestibility and metabolisable energy of feeds through direct interaction with proteins and carbohydrates from both exogenous and endogenous sources. In an experiment, sixty-four, 10-week-old broiler cockerels, in eight treatments of eight replicates, were fed warm glucose solution (30 g/50 ml, 40 °C) or water (50 ml) alone or with TA solution (3, 6, and 10 g/15 ml) by an oral gavage. Excreta voided from each bird following precision feeding, was collected quantitatively, for 48 h. The estimated amino acids in the excreta by HPLC showed an increase (P<0.001) in the endogenous losses of individual amino acids by tannic acid. The amino acids most affected were methionine (4.6-fold), histidine (4.8-fold), and lysine (4.3-fold) and the least affected amino acids were threonine (2.4-fold), cysteine (2.5-fold), and valine (2.4-fold) when the maximum amount of TA (10 g) was administered. There was no difference in the slope of the excretion of individual amino acids between glucose- and water-fed birds when the amount of TA increased. The possible mechanisms by which tannic acid increased linearly the loss of individual amino acids with endogenous origin are discussed.

Liquid chromatography-based determination of urinary free and total N(epsilon)-(carboxymethyl)lysine excretion in normal and diabetic subjects

We propose a specific, reproducible and sensitive HPLC method for the determination of N(epsilon)-(carboxymethyl)lysine (CML) excreted in urine. Total CML was measured in acid hydrolysates of urine samples, while free CML was measured in acetonitrile-deproteinised urine samples using a RP-HPLC method with ortho-phtaldialdehyde (OPA)-derivatisation and fluorescence detection suited for automation. We compared the CML excretion of 51 non-proteinuric patients with diabetes mellitus (DM) (age 57±14 years, HbA1c 8.0±1.8%) to 42 non-diabetic controls (C) (age 45±17 years). The urinary excretion of total CML in diabetic patients was increased by ∼30% (DM: 0.58±0.21; C: 0.45±0.14 μmol/mmol creatinine; P<0.001). While urinary excretion of free CML was not significantly different, excretion of bound CML was increased (DM: 0.36±0.17; C: 0.27±0.14; P<0.05) in diabetic patients. CML excretion was correlated with protein and albumin excretion, but did not correlate with HbA1c, duration of DM or diabetic complications such as neuropathy or retinopathy. Furthermore, no age-dependent change of total CML excretion was found, while free CML excretion was lower in younger subjects. The specific and sensitive determination of CML by RP-HPLC of its OPA-derivative is well suited for automation and better than that of less defined glycoxidation products (AGEs).

Medical and surgical therapy of the cystine stone patient.

Cystinuria is an inherited defect in renal tubular and intestinal transport of dibasic amino acids that results in elevated urinary excretion of cystine, ornithine, lysine, and arginine. The only clinical manifestation of this disease is the development of urinary tract cystine calculi. Cystinuric patients suffer recurrent stone episodes, requiring an aggressive multi-modal approach to management. This article reviews the results of medical prevention regimens, the role of minimally invasive urologic intervention, and the recent insights into the complex genetic basis for the varied cystinuric phenotypes.

Availability and utilisation of free lysine in rainbow trout ( Oncorhynchus mykiss) : 1. Effect of dietary crude protein level

It was investigated whether the availability and utilisation of free lysine are affected by crude protein concentration in rainbow trout diets. Wheat gluten and free amino acids were used as sole sources of amino acids to formulate two basal diets differing in crude protein (350 and 550 g/kg dry matter) but equal in lysine concentration (9 g/kg dry matter). Lysine was supplemented at the expense of l-alanine to achieve further lysine levels of 13 and 17 g/kg dry matter. l-lysine·HCl was used as the supplemental lysine source at both crude protein levels. Diets were offered to three groups of 20 trout each over a period of 56 days. Amount of feed was restricted to about 16 g dry matter/kg BW daily in order to allow for an equal and complete intake in all groups. Trout had an initial body weight of 50 g/fish. Digestibility was determined using acid-insoluble ash as a marker. Faecal samples were collected via a sedimentation system attached to each tank. Retention was determined by comparative whole body analysis. Faecal lysine excretion was unaffected by lysine supplementation irrespective of dietary crude protein level. Hence, l-lysine·HCl can be regarded 100% available to trout. In spite of comparable levels of DE intake, growth and gain/feed ratio increased with lysine supplementation. In gained body mass, protein concentration was increased and lipid concentration was decreased with increasing lysine supplementation. Amino acid pattern of gained body protein was changed by lysine supplementation as well. Lysine utilisation, determined as Δretained/Δdigested×100, was 55% on the low and 71% on the high dietary crude protein levels, respectively. This difference was close to significance, as judged by comparative regression analysis ( P=0.11). Dose–response studies with a wider range in lysine concentration at different crude protein levels are necessary to identify the consequences regarding lysine allowances for practical type trout diets.

Availability and utilisation of free lysine in rainbow trout ( Oncorhynchus mykiss) : 2. Comparison of l-lysine·HCl and l-lysine sulphate

A basal diet, aiming at a low lysine concentration (9 g/kg DM), was formulated with wheat gluten as the sole source of protein. Lysine concentration was increased in two steps of approximately 4 g/kg DM each by use of either l-lysine·HCl or l-lysine sulphate. Each diet was fed at a restricted rate to three groups of 20 rainbow trout for 56 days. Faecal samples were collected via a sedimentation system and availability was determined using acid-insoluble ash as a marker. Retention was measured via comparative whole body analysis. Faecal lysine excretion was not affected by lysine supplementation. Body weight gain and gain/feed ratio, as well as the composition of body weight gain, were significantly affected by dietary lysine concentration but not by lysine source. The utilisation of supplemental lysine, calculated as Δretained/Δdigested×100, was, on average, 68% and was not significantly different between the two lysine sources ( P=0.62). It is concluded that both lysine sources are equally available to rainbow trout.

Effect of lysine infusion on urea cycle in lysinuric protein intolerance

Poor intestinal absorption and excessive renal loss of dibasic amino acids result in low plasma concentrations in patients with lysinuric protein intolerance (LPI). Arginine and ornithine deficiency impair the function of the urea cycle and cause hyperammonemia after protein intake, while chronic lysine deficiency may cause growth failure and lead to reduced bone density in such patients. Since high lysine concentrations inhibit several enzymes of the urea cycle in the liver, lysine supplementation may induce hyperammonemia in LPI. We thus studied how LPI patients tolerate high plasma lysine by intravenous (IV) infusion of 3.3 mmol/kg lysine hydrochloride over 90 minutes in 6 adult patients and 4 healthy controls. The plasma lysine concentration (mean +/- SD, range) peaked in the patients (9,114 +/- 1,864, 7,156 to 12,044 micromol/L) and controls (10,185 +/- 2,253, 7,714to 13,122 micromol/L) at 90 minutes. Urinary lysine excretion peaked in the second 2-hour urine collection in the patients (4,582 +/- 1,276, 3,018 to 6,315 micromol/m2 body surface area per hour) and in the first 2-hour collection in the controls (5,373 +/- 1,766, 3,551 to 7,286 micromol/m2/h). Two patients had mild nausea but no hyperammonemia and one patient had moderate hyperammonemia (peak, 112 micromol/L) at the end of the infusion. Orotic acid excretion increased in 2 subjects with a peak excretion rate of 33 and 251 micromol/m2/h in the third 2-hour collection after starting the load. All other subjects remained asymptomatic and showed no change in plasma ammonia or urinary orotic acid excretion. We thus conclude that an acute increase in plasma lysine caused minimal clinical or biochemical untoward effects in patients with LPI. Moderate increases in plasma lysine after low-dose oral supplementation with lysine or well-absorbed lysine derivatives are probably well tolerated in LPI.

Reference values of urinary excretion of cystine and dibasic aminoacids: classification of patients with cystinuria in the Valencian Community, Spain

Objective: Cystinuria is an autosomal-recessive disorder of the kidneys and small intestine affecting a luminal transport mechanism shared by cystine, ornithine, arginine, and lysine. Three different types of cystinuria can be distinguished according to the excretion of these amino acids in urine samples. We propose cutoff values from our population as references and we present a classification of cystinuric patients using quantitative amino acid chromatography in first morning urine samples. Design and methods: A random sample of forty healthy subjects belonging to general population of the Valencian Community were selected as control subjects. Cystine, lysine, arginine, and ornithine were quantified by reverse-phase HPLC. Seventy-two subjects, diagnosed previously as cystinuric by the cyanide-nitroprusside test were classified. Probands excreting more than 113.12 μmol cystine per mmol of creatinine ( i.e., 1,000 μmol cystine per gram of creatinine) were classified as homozygotes. Parents of homozygotes in whom excretion of amino acids were normal were classified as heterozygotes type I. Those probands showing the excretion of at least one amino acid and the sum of urinary cistine plus the basic amino acids higher than the corresponding references ranges in our population were classified as heterozygotes type II or type III (heterozygotes non-type I). Results: We identified 24 homozygotes, 39 non-type I heterozygotes and 3 type I heterozygotes. The remaining 6 probands could not be classified. Means for cystine, lysine, arginine ornithine and their sum in homozygotes and heterozygotes non-type I were significantly ( p < 0.001) in excess of the respective reference ranges. Moreover, means values in homozygotes were statiscally different ( p < 0.001) from heterozygotes non-type I. Conclusion: Urinary excretion of cystine per mmol creatinine allow us to distinguish heterozygotes from homozygotes. However, the best discriminator to distinguish non-type I heterozygotes from normal population might be the excretion of lysine per mmol creatinine. Additional studies including characterization of appropiate haplotypes should be carried out for a more precise identification of types of cystinuria.

Lysine production byBrevibacterium linens and its mutants: Activities and regulation of enzymes of the lysine biosynthetic pathway.

Activity and regulation of key enzymes of the lysine biosynthetic pathway were investigated inBrevibacterium linens, a natural excretor of lysine, its lysine-overproducing homoserine auxotroph (Hom(-1)) and its auxotrophic and multianalogue-resistant high-yielding mutant (AEC NV 20(r)50). The activity of aspartate kinase (AK) and aspartaldehydate dehydrogenase (AD) was maximum during the mid-exponential phase of growth and decreased therafter. The mutants showed 10 and 20% more activity of AK and AD than the wild-type lysine excretor.B. linens (natural excretor) has a single AK and AD repressed and inhibited bivalently by lysine and threonine. Lysine slightly repressed and inhibited dihydrodipicolinate synthase (DS) and diaminopimelate decarboxylase (DD) of the wild type and of the mutant Hom(-1). The mutant AEC NV 20(r)50 showed DS and DD to be insensitive to lysine inhibition and repression. Persistence of a major part of the maximal activity of these enzymes during the late stationary phase of growth allowed prolonged synthesis and excretion of lysine. Stepwise addition of resistance to the different analogues of lysine in the mutant AEC NV20(r)50 resulted in an increase of enzyme activity and reduced repressibilities of enzymes that contributed to the high yield of lysine.

Fed-batch production of L-lysine by Corynebacterium glutamicum

The growth rate, glucose consumption rate, and production rate of an L-lysine producing Corynebacterium glutamicum mutant were studied in batch and fed-batch cultures. In batch fermentation, higher L-lysine productivity (1.93 g l −1 h −1) and L-lysine yield of 0.70 g g −1 were obtained with lower lysine excretion (34 g l −1). A strong enhancement of L-lysine production (110.6 g l −1) was obtained in fed-batch culture, whereas the kinetic parameters remained lower than those observed in batch cultures. In both culture techniques high L-lysine productivity and L-lysine yield occurred at the lower growth rate ranging from 0.02 h −1. Enzymatic analysis revealed that the higher L-lysine production could be obtained under a higher phosphoenolpyruvate carboxylase/citrate synthase enzyme activity ratio to avoid biomass production at the expense of L-lysine. This is a direct proof of the important role of both phosphoenolpyruvate carboxylase activity and the composition of the growth medium on the L-lysine overproduction phase.