Determination of leucine and α-ketoisocaproic acid concentrations and specific activity in plasma and leucine specific activities in proteins using high-performance liquid chromatography

Horber, Fritz F.; Kahl, Jane; Lecavalier, L.; Krom, Bethany; Haymond, Morey W.

doi:10.1016/s0378-4347(00)82611-0

Cited by 58 publications

(43 citation statements)

References 9 publications

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“…Plasma [ 13 C] and [ 2 H5]glutamine enrichments were determined by selected ion monitoring GCMS (Hewlett-Packard MSD 5971) as described previously (10). Plasma [ 14 C]KIC specific activity was determined by HPLC (on a Spectraphysics SP8800 instrument equipped with a Pharmacia LKB Frac 200 fraction collector) as described previously (23). The 14 C-specific activity of KIC fractions and breath CO2 was measured on a Beckman LS6500 scintillation counter.…”

Section: Methodsmentioning

confidence: 99%

Corticosteroids increase glutamine utilization in human splanchnic bed

Thibault

Welch²,

Mauras³

et al. 2008

American Journal of Physiology-Gastrointestinal and Liver Physi

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Thibault R, Welch S, Mauras N, Sager B, Altomare A, Haymond M, Darmaun D. Corticosteroids increase glutamine utilization in human splanchnic bed. Am J Physiol Gastrointest Liver Physiol 294: G548-G553, 2008. First published December 27, 2007 doi:10.1152/ajpgi.00461.2007.-Glutamine is the most abundant amino acid in the body and is extensively taken up in gut and liver in healthy humans. To determine whether glucocorticosteroids alter splanchnic glutamine metabolism, the effect of prednisone was assessed in healthy volunteers using isotope tracer methods. Two groups of healthy adults received 5-h intravenous infusions of L-[1-14 C]leucine and L-[ 2 H5]glutamine, along with q. 20 min oral sips of tracer doses of L-[1-13 C]glutamine in the fasting state, either 1) at baseline (control group; n ϭ 6) or 2) after a 6-day course of 0.8 mg ⅐ kg Ϫ1 ⅐ day Ϫ1 prednisone (prednisone group; n ϭ 8). Leucine and glutamine appearance rates (Ra) were determined from plasma [1-14 C]ketoisocaproate and [ 2 H5]glutamine, respectively, and leucine and glutamine oxidation from breath 14 CO2 and 13 CO2, respectively. Splanchnic glutamine extraction was estimated by the fraction of orally administered [ 13 C]glutamine that failed to appear into systemic blood. Prednisone treatment 1) did not affect leucine Ra or leucine oxidation; 2) increased plasma glutamine Ra, mostly owing to enhanced glutamine de novo synthesis (medians Ϯ interquartiles, 412 Ϯ 61 vs. 280 Ϯ 190 mol ⅐ kg Ϫ1 ⅐ h Ϫ1 , P ϭ 0.003); and 3) increased the fraction of orally administered glutamine undergoing extraction in the splanchnic territory (means Ϯ SE 64 Ϯ 6 vs. 42 Ϯ 12%, P Ͻ 0.05), without any change in the fraction of glutamine oxidized (means Ϯ SE, 75 Ϯ 4 vs. 77 Ϯ 4%, not significant). We conclude that high-dose glucocorticosteroids increase in splanchnic bed the glutamine requirements. The role of such changes in patients receiving chronic corticoid treatment for inflammatory diseases or suffering from severe illness remains to be determined. nutrition; protein metabolism; stable isotopes; stress; gut GLUTAMINE PLAYS AN IMPORTANT role in the maintenance of intestinal function (39,43,46). Glutamine indeed is both a preferred fuel (9, 47) for enterocytes and a precursor for the intestinal synthesis of purines, pyrimidines (9), and glutathione (37), and it may regulate gut protein synthesis (21,30,32). In addition, glutamine is a major donor of carbon for gluconeogenesis (19). This explains why glutamine is extensively extracted in the splanchnic tissues in several animal species (39,47).In humans, the concomitant infusion of two tracers of glutamine (one infused intravenously, the other one enterally) can be used to assess the extraction of glutamine by the splanchnic bed. By using this technique, ϳ50 to 75% of enterally administered glutamine undergoes first-pass extraction within the splanchnic bed, presumably by the gut and liver, in healthy humans (4, 17, 18, 34). Additionally, the major fate of splanchnic glutamine after enteral administration was oxidation, ...

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Section: Methodsmentioning

confidence: 99%

Corticosteroids increase glutamine utilization in human splanchnic bed

Thibault

Welch²,

Mauras³

et al. 2008

American Journal of Physiology-Gastrointestinal and Liver Physi

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“…Analytical determinations Leucine and KIC were extracted from plasma samples as previously described [13]. Enrichments and concentrations of plasma leucine and KIC were determined on their t-butyldimethylsilyl derivatives using gas chromatography-mass spectrometry in electron impact ionisation mode (GC8000, MS Voyager Finningan; ThermoQuest Italia, Milan, Italy), monitoring the ions 302 and 305 for leucine and 301 and 304 for KIC [14].…”

Section: Methodsmentioning

confidence: 99%

Effects of dietary protein restriction on albumin and fibrinogen synthesis in macroalbuminuric type 2 diabetic patients

et al. 2007

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Aims/hypothesis Diabetic nephropathy is associated with hypoalbuminaemia and hyperfibrinogenaemia. A lowprotein diet has been recommended in patients with diabetic nephropathy, but its effects on albumin and fibrinogen synthesis are unknown. Methods We compared the effects of a normal (NPD; 1.38± 0.08 g kg −1 day −1 ) or low (LPD; 0.81±0.04 g kg −1 day −1 ) -protein diet on endogenous leucine flux (ELF), albumin and fibrinogen synthesis (L-[5,5,5,-2 H 3 ]leucine infusion), and markers of inflammation in nine type 2 diabetic patients with macroalbuminuria. Six healthy participants on NPD served as control participants. Results In comparison with healthy participants, type 2 diabetic patients on an NPD had similar ELF, reduced serum albumin (38±1.1 vs 42±0.8 g/l; p<0.05), similar fractional synthesis rates (FSR) and absolute synthesis rates (ASR) of albumin, and both increased plasma fibrinogen concentration [10.7±0.6 vs 7.2±0.5 μmol/l (3.64±0.22 vs 2.45±0.18 g/l); p<0.05] and fibrinogen ASR [11.03±1.17 vs 6.0±1.8 μmol 1.73 m −2 day −1 (3.7±0.4 vs 1.9±0.3 g 1.73 m −2 day −1 ); p< 0.01]. After LPD, type 2 diabetic patients had the following changes in comparison with NPD: reduced proteinuria (2.74±0.4 vs 4.51±0.8 g/day; p<0.05), ELF (1.93±0.08 vs 2.11±0.08 μmol kg −1 min −1 ; p<0.05) and total fibrinogen pool; increased serum albumin (42±1 vs 38±1 g/l; p<0.01) and albumin ASR (14.1±1 vs 9.9±1 g 1.73 m −2 day −1 ; p< 0.05); and reduced plasma IL-6 levels, which were correlated with albumin ASR (r=−0.749; p<0.05). Conclusions/interpretation LPD in type 2 diabetic patients with diabetic nephropathy reduces low-grade inflammatory state, proteinuria, albuminuria, whole-body proteolysis and ASR of fibrinogen, while increasing albumin FSR, ASR and serum concentration. ISRCTN ID no: CCT-NAPN-16911Keywords Absolute synthesis . Albumin . Diabetic nephropathy . Fibrinogen . Fractional synthesis . Leucine . Low-grade inflammation . Low-protein diet . Macroalbuminuria . Type 2 diabetes Abbreviations ASR absolute synthesis rate CRP C-reactive protein ELF endogenous leucine flux FSR fractional synthesis rate KIC α-ketoisocaproic acid LPD low-protein diet Diabetologia (2008) 51:21-28

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“…Plasma glucose, albumin, triglyceride, total and HDL cholesterol, creatinine, CRP (not the high-sensitivity assay) and urinary albumin concentrations were all determined by standard laboratory methods. Albumin and fibrinogen were isolated from plasma and hydrolysed using standard and validated methods as previously described [19,20,24]. Plasma leucine and α-ketoisocaproate (KIC) concentrations and SA, as well as leucine SA in the albumin and fibrinogen hydrolysate, were determined by HPLC (Waters Spa Italia, Milano, Italy) [24].…”

Section: Biochemical Determinations Calculations and Statistical Anamentioning

confidence: 99%

“…Albumin and fibrinogen were isolated from plasma and hydrolysed using standard and validated methods as previously described [19,20,24]. Plasma leucine and α-ketoisocaproate (KIC) concentrations and SA, as well as leucine SA in the albumin and fibrinogen hydrolysate, were determined by HPLC (Waters Spa Italia, Milano, Italy) [24]. Insulin, glucagon and C-peptide concentrations were determined by radioimmunoassay as described elsewhere [20,21].…”

Section: Biochemical Determinations Calculations and Statistical Anamentioning

confidence: 99%

“…Plasma amino acid concentrations were measured with a Beckman Amino Acid Analyzer (Beckman Instruments, Palo Alto, CA, USA). The albumin and fibrinogen fractional synthesis rates (FSR), expressed as a percentage of the circulating pool of each per day, were calculated from the increment of the SA values of leucine incorporated into either protein with respect to time, and divided by average plasma KIC SA, between 120 min and the end of the infusion (Table 2), using a standard linear relationship [13,18,20,24,25]. Within this interval, the chosen plasma precursor pool (i.e.…”

Section: Biochemical Determinations Calculations and Statistical Anamentioning

confidence: 99%

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Diabetic nephropathy is associated with increased albumin and fibrinogen production in patients with type 2 diabetes

et al. 2006

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Aims/hypothesis: Hyperfibrinogenaemia and albuminuria are cardiovascular risk factors, often coexisting in diabetic and non-diabetic people. Albuminuria in turn is associated with a compensatory albumin overproduction in non-diabetic patients. It is not known whether the presence of albuminuria in patients with type 2 diabetes mellitus is associated with greater albumin and fibrinogen production rates than in normoalbuminuric patients. Subjects, materials, and methods: Using leucine isotope methods, we measured fractional and absolute synthesis rates (FSR, ASR) of albumin and fibrinogen in post-absorptive type 2 diabetic patients with either normal (n=11) or increased (n=10) urinary albumin excretion. Results: In albuminuric patients, albumin FSR (16.2± 1.5%/day) and ASR (20.5±1.9 g/day) were greater (p<0.02 and p<0.05, respectively) than in normoalbuminuric patients (FSR=11.5±1.1%/day; ASR=15.7±1.2 g/day). Fibrinogen FSR was similar between patients with normal and increased albumin excretion, but concentration, the circulating pool and ASR of fibrinogen were 40 to 50% greater (p<0.035) in patients with albuminuria. Albuminuria was positively correlated with albumin ASR, with fibrinogen concentration, the fibrinogen pool and ASR, whereas albumin synthesis was inversely correlated with calculated oncotic pressure. Conclusions/interpretation: Synthesis of albumin and fibrinogen is upregulated in type 2 diabetic patients with increased urinary albumin excretion. Albuminuria is associated with enhanced fibrinogen and albumin synthesis.

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Determination of leucine and α-ketoisocaproic acid concentrations and specific activity in plasma and leucine specific activities in proteins using high-performance liquid chromatography

Cited by 58 publications

References 9 publications

Corticosteroids increase glutamine utilization in human splanchnic bed

Corticosteroids increase glutamine utilization in human splanchnic bed

Effects of dietary protein restriction on albumin and fibrinogen synthesis in macroalbuminuric type 2 diabetic patients

Diabetic nephropathy is associated with increased albumin and fibrinogen production in patients with type 2 diabetes

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