Transcription factor 7-like 2 gene links increased in vivo insulin synthesis to type 2 diabetes.

Transcription factor 7-like 2 (TCF7L2) is the main susceptibility gene for type 2 diabetes, primarily through impairing the insulin secretion by pancreatic ß cells. However, the exact in vivo mechanisms remain poorly understood. We performed a family study and determined if the T risk allele of the rs7903146 in the TCF7L2 gene increases the risk of type 2 diabetes based on real-time stable isotope measurements of insulin synthesis during an Oral Glucose Tolerance Test. In addition, we performed oral minimal model (OMM) analyses to assess insulin sensitivity and ß cell function indices. Compared to unaffected relatives, individuals with type 2 diabetes had lower OMM indices and a higher level of insulin synthesis. We found a T allele-dosage effect on insulin synthesis and on glucose tolerance status, therefore insulin synthesis was higher among T-allele carriers with type 2 diabetes than in wild-type individuals. These results suggest that hyperinsulinemia is not only an adaptation to insulin resistance, but also a direct cause of type 2 diabetes.

Peritoneal protein losses and cytokine generation in automated peritoneal dialysis with combined amino acids and glucose solutions.

OBJECTIVES: Protein-energy malnutrition as a consequence of deficient protein intake frequently occurs in peritoneal dialysis (PD) patients. Previously, we showed that peritoneal dialysate containing a mixture of amino acids (AA) and glucose has anabolic effects. However AA-dialysate has been reported to increase intraperitoneal protein and AA losses and the release of proinflammatory cytokines (interleukine-6 (IL-6) and tumor necrosis factor alpha (TNFalpha)). We investigated the effect of AA plus glucose (AAG) solutions on peritoneal protein losses and cytokine generation. METHODS: In 6 patients on standard automated peritoneal dialysis (APD) 12 APD sessions of 6 cycles each were performed during the night using dialysate containing 1.1% AA plus glucose or glucose alone as control. Protein losses and TNFalpha and IL-6 concentrations were measured in dialysates separately collected from nightly cycling and daytime dwell. RESULTS: The 24 hour-protein losses with AAG (median 6.7 g, range 4.7-9.4 g) were similar to control dialysate (median 6.0 g, range 4.2-9.2 g). Daytime dialysate IL-6 levels were higher after nightly AAG dialysis than after control dialysis (142 pg/ml and 82 pg/ml, respectively, P<.05). TNFalpha concentrations were very low. CONCLUSION: Nightly APD with amino acids containing dialysate was associated with an increase in peritoneal IL-6 generation during the day. The addition of AA to standard glucose dialysis solutions did not induce a significant increase of peritoneal protein losses.

A stable isotope method for in vivo assessment of human insulin synthesis and secretion.

AIMS: In vitro, beta cells immediately secrete stored but readily releasable insulin in response to a rise of glucose. During a prolonged insulin response, this is followed by newly synthesized insulin. Our aim was to develop an in vivo test to determine the ratio between readily available and newly synthesized insulin after a stimulus in humans by labelling newly synthesized insulin. METHODS: A stable isotope tracer of 1.0 g 13C leucine with C-peptide as target peptide was administered 45 min prior to 75 g glucose load of a frequently blood sampled 210-min oral glucose tolerance test (OGTT). Our OGTT also encompassed collection of urine, which has a high content of C-peptide. Prior, the optimal conditions under which the tracer 13C leucine was administered for enrichment of (pre) proinsulin were established. Also, techniques to obtain urinary C-peptide under highly purified circumstances were set up. Our main outcome measure was the stable isotope enrichment of de novo C-peptide, which we related to early plasma insulin and glucose AUC. Twelve healthy Caucasian individuals (M4F8, age 41.8 ± 2.3, BMI 28.3 ± 1.7) with normal glucose tolerance underwent our OGTT. RESULTS: We found that during a 75-g OGTT, newly synthesized insulin contributed approximately 20 % of total insulin secretion. The pattern of isotope enrichment obtained by collecting multiple urine voids was suggestive that the newly synthesized insulin contributes to the late phase of insulin secretion. De novo C-peptide correlated negatively with both early plasma insulin AUC (r = -0.629, P = 0.028) and early plasma glucose AUC (r = -0.605, P = 0.037). CONCLUSIONS: With stable isotope technique added to OGTT, we were able to measure newly synthesized insulin in healthy individuals. This new technique holds the promise that it is feasible to develop a direct in vivo beta cell function test.

Post-glucose-load urinary C-peptide and glucose concentration obtained during OGTT do not affect oral minimal model-based plasma indices.

The purpose of this study was to investigate how renal loss of both C-peptide and glucose during oral glucose tolerance test (OGTT) relate to and affect plasma-derived oral minimal model (OMM) indices. All individuals were recruited during family screening between August 2007 and January 2011 and underwent a 3.5-h OGTT, collecting nine plasma samples and urine during OGTT. We obtained the following three subgroups: normoglycemic, at risk, and T2D. We recruited South Asian and Caucasian families, and we report separate analyses if differences occurred. Plasma glucose, insulin, and C-peptide concentrations were analyzed as AUCs during OGTT, OMM estimate of renal C-peptide secretion, and OMM beta-cell and insulin sensitivity indices were calculated to obtain disposition indices. Post-glucose load glucose and C-peptide in urine were measured and related to plasma-based indices. Urinary glucose corresponded well with plasma glucose AUC (Cau r = 0.64, P < 0.01; SA r = 0.69, P < 0.01), S I (Cau r = -0.51, P < 0.01; SA r = -0.41, P < 0.01), Φ dynamic (Cau r = -0.41, P < 0.01; SA r = -0.57, P < 0.01), and Φ oral (Cau r = -0.61, P < 0.01; SA r = -0.73, P < 0.01). Urinary C-peptide corresponded well to plasma C-peptide AUC (Cau r = 0.45, P < 0.01; SA r = 0.33, P < 0.05) and OMM estimate of renal C-peptide secretion (r = 0.42, P < 0.01). In general, glucose excretion plasma threshold for the presence of glucose in urine was ~10-10.5 mmol L(-1) in non-T2D individuals, but not measurable in T2D individuals. Renal glucose secretion during OGTT did not influence OMM indices in general nor in T2D patients (renal clearance range 0-2.1 %, with median 0.2 % of plasma glucose AUC). C-indices of urinary glucose to detect various stages of glucose intolerance were excellent (Cau 0.83-0.98; SA 0.75-0.89). The limited role of renal glucose secretion validates the neglecting of urinary glucose secretion in kinetic models of glucose homeostasis using plasma glucose concentrations. Both C-peptide and glucose in urine collected during OGTT might be used as non-invasive measures for endogenous insulin secretion and glucose tolerance state.

Discriminative Ability of Plasma Branched-Chain Amino Acid Levels for Glucose Intolerance in Families At Risk for Type 2 Diabetes.

BACKGROUND: Insulin resistance and glucose intolerance have been associated with increased plasma levels of branched-chain amino acids (BCAA). BCAA levels do not predict T2DM in the population. We determined the discriminative ability of fasting BCAA levels for glucose intolerance in nondiabetic relatives of patients with T2DM of two different ethnicities. METHODS: Based on oral glucose tolerance test (OGTT), first-degree relatives of patients with T2DM were categorized as normal glucose tolerance, prediabetes, or T2DM. Included were 34, 12, and 18 Caucasian and 22, 12, and 23 Asian Indian participants, respectively. BCAA levels were measured in fasting plasma together with alanine, phenylalanine, and tyrosine. Insulin sensitivity and beta-cell function were assessed by indices derived from an extended OGTT and their relationship with plasma BCAA levels was assessed in multivariate regression analysis. The value of the amino acids for discriminating prediabetes among nondiabetic family members was determined with the area under the curve of receiver-operated characteristics (c-index). RESULTS: BCAA levels were higher in diabetic than in normoglycemic family members in the Caucasians (P = 0.001) but not in the Asian Indians. In both groups, BCAA levels were associated with waist-hip ratio (ß = 0.31; P = 0.03 and ß = 0.42; P = 0.001, respectively) but not with indices of insulin sensitivity or beta-cell function. The c-index of BCAA for discriminating prediabetes among nondiabetic participants was 0.83 and 0.74 in Caucasians and Asian Indians, respectively, which increased to 0.84 and 0.79 by also including the other amino acids. The c-index of fasting glucose for discriminating prediabetes increased from 0.91 to 0.92 in Caucasians and 0.85 to 0.97 (P = 0.04) in Asian Indians by inclusion of BCAA+alanine, phenylalanine, and tyrosine. CONCLUSIONS: Adding fasting plasma BCAA levels, combined with phenylalanine, tyrosine and alanine to fasting glucose improved discriminative ability for the prediabetic state within Asian Indian families at risk for T2DM. BCAA levels may serve as biomarkers for early development of glucose intolerance in these families.

Conversion of octanoic acid into long-chain saturated fatty acids in premature infants fed a formula containing medium-chain triglycerides.

A large number of very-low-birth weight infants are fed formulas containing medium-chain triglycerides (MCT) to enhance fat and calcium absorption. Studies are available on the intestinal absorption of MCT, which is nearly complete, but uncertainties exist on the metabolic fate of octanoic acid, the major component of MCT. Oxidation accounts for approximately 50% of the dietary intake, and losses as dicarboxylic acids in the urine are negligible. Since storage in adipose tissue is limited, conversion into long-chain fatty acids (LCFA) is likely to be an important route. To study the nonoxidative metabolism of MCT, six preterm infants fed a standard premature formula containing 38 weight% (wt%) MCT (54 mol% medium-chain fatty acids (MCFA), of which 35 mol% is octanoic acid) were studied at 4 weeks of age, when on full oral intake and receiving on average 130 kcal/kg/d. The study consisted of an oral primed constant-rate infusion of [13C]-octanoate and the measurement of the 13C enrichment of individual fatty acids in plasma triglycerides (TG) by a highly sensitive on-line combustion method using gas chromatography-isotope ratio mass spectrometry (GC-IRMS). We observed a significant incorporation of the dietary [13C]-octanoic acid in plasma TG (10.0% +/- 4.5% of the enrichment of the diet). A noticeable incorporation of the label was detected in myristic and palmitic acids (4.6% +/- 2.5% and 7.8% +/- 4.1% of the octanoic enrichment of the diet). The absolute amount of the fatty acids was studied with conventional GC, and the plasma TG fatty acid profile differed markedly from the diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipolysis and lipid oxidation in weight-losing cancer patients and healthy subjects.

Increased lipolysis has been suggested as one of the possible mechanisms underlying cancer cachexia. The study aim was to assess whether lipolysis is increased in weight-losing cancer patients, considering their differences in food intake and body composition. Sixteen healthy subjects and 18 cancer patients with different tumor types and a weight loss of at least 5% in the previous 6 months were included in the study. Food intake was recorded for 4 days. After an overnight fast, [1,1,2,3,3-2H5]glycerol was infused to determine the rate of appearance (Ra) of glycerol as a measure of whole-body lipolysis, and [1-13C]palmitic acid was infused to determine the Ra of palmitate as a measure of adipocyte fatty acid release. Palmitate oxidation was determined by measuring 13CO2 enrichment in breath samples, and body composition was measured by bioelectrical impedance analysis. After adjustment for energy intake, whole-body lipolysis was significantly higher in cancer patients versus healthy subjects (6.46 +/- 0.63 and 4.67 +/- 0.46 micromol/kg +/- min, respectively, P < .05). The difference in adipocyte fatty acid release did not reach statistical significance. The rate of palmitate oxidation was also significantly higher in patients than in healthy subjects (1.15 +/- 0.10 and 0.93 +/- 0.07 )micromol/kg x min, respectively, P < .05). No differences in body composition were observed between groups. In conclusion, whole-body lipolysis (as measured by the Ra of glycerol) and palmitate oxidation were elevated in weight-losing cancer patients, but fatty acid release was not significantly different.

Eicosapentaenoic acid ethyl ester supplementation in cachectic cancer patients and healthy subjects: effects on lipolysis and lipid oxidation.

BACKGROUND & AIMS: Recent reports suggest that weight loss in cachectic cancer patients may be inhibited by supplementation of the n-3 fatty acid eicosapentaenoic acid (20:5n-3; EPA), presumably due to inhibition of lipolysis. The aim of the present double-blind, randomized trial was to assess whether short-term oral EPA ethyl ester (EE) supplementation inhibits lipolysis and lipid oxidation in weight-losing cancer patients and in healthy subjects. METHODS: Seventeen weight-losing, cancer patients of different tumor types, and 16 healthy subjects were randomized to receive EPA-EE (6 g/d) or placebo (oleic acid (OA)-EE; 6 g/d) for seven days. At baseline (day 0) and during supplementation (days 2 and 7) whole-body lipolysis and palmitic acid release were measured in the overnight fasting state using [1, 1, 2, 3, 3-(2)H(5)]glycerol and [1-(13)C]palmitic acid. Palmitate oxidation was determined by measuring(13)CO(2)enrichment in expired breath. RESULTS: No significant effects of EPA-EE on whole-body lipolysis, palmitic acid release, or palmitate oxidation were detected in cancer patients nor in healthy subjects in comparison with OA-EE. EPA-EE supplementation reduced plasma-free fatty acid and triacylglycerol concentrations significantly in healthy subjects but not in cancer patients. CONCLUSION: We conclude that supplementation of EPA-EE does not significantly inhibit lipolysis or lipid oxidation in weight-losing cancer patients or in healthy subjects during short-term supplementation when using OA-EE as a placebo supplement.

Influence of the 13C-enrichment of the habitual diet on a 13CO2 breath test used as an index of liver glycogen oxidation: a validation study in western Europe and Africa.

A diet containing naturally 13C-enriched carbohydrate combined with a 13CO2 breath-test analysis can be used to monitor liver glycogen oxidation in persons used to a diet low in 13C, e.g., the Western European diet. In this study, we evaluated this test principle further by changing the way we label the glycogen pool. The 13C enrichment of exhaled CO2 was studied in two groups, one in Europe and one in Africa. The European group (n = 12) was accustomed to a diet low in 13C, and they went on a 13C-enriched study diet to identify liver glycogen. The African group (n = 6) was accustomed to a diet naturally high in 13C, and they went on a diet low in 13C. The basal 13C abundance in exhaled CO2 was higher in the African group (1.0879 At%; atmospheric 1.1 atom percent) than in the European group (1.0821 At%). During the study period, the parameters for liver glycogen oxidation--the 13CO2 enrichment plateau, the plateau duration, and the return to baseline time--did not differ between groups. The abundance of 13CO2 in exhaled CO2 over time in the two groups was similar but inverse. This study confirms the use of a 13CO2 breath test to monitor liver glycogen oxidation and demonstrates how to use such a test in persons accustomed to a diet high in 13C.

The 13CO2 breath test for liver glycogen oxidation after 3-day labeling of the liver with a naturally 13C-enriched diet.

OBJECTIVE: When naturally (13)C-enriched carbohydrate is used to label hepatic glycogen, (13)C-liver glycogen oxidation can be monitored subsequently by measuring the (13)C enrichment of breath CO(2) during a sedentary fast. In our previous breath test studies, we used a 1-d labeling protocol to enrich liver glycogen. Others found that after 3 d of labeling the liver glycogen (13)C enrichment is identical to the dietary carbohydrate (13)C enrichment. METHODS: We compared a diet protocol in which naturally (13)C-enriched carbohydrate was given for 3 d before the breath test with our previously applied 1-d labeling design. The (13)CO(2) breath test was combined with indirect calorimetry. The results were compared with those from our previous studies. In addition, we compared liver glycogen oxidation rates with those from our present technique and different techniques as used in other published studies. RESULTS: Six healthy volunteers were included in this study. The (13)C enrichment of breath CO(2) at plateau excretion level did not differ after 1 or 3 d on a labeling diet. However, the end of plateau time tended to be later after the 3-d diet, 14.3 h versus 12.5 to 13.5 h postprandially in the 1-d labeling studies. Also, the return to baseline time was later in the 3-d study, at 25.8 h versus 19.0 to 23.2 h postprandially after 1 d of labeling. The liver glycogen oxidation rate was similar in both techniques until 17 h postprandially. After this time the 3-d labeling protocol showed a higher level of liver glycogen oxidation. CONCLUSION: The results indicated that the labeling of liver glycogen is slightly less complete after 1 d on a (13)C-enriched diet as compared with 3-d labeling. Our (13)C breath test results compared rather well with studies from the literature using the (13)C-NMR technique, the D(2)O technique, or the (13)CO(2) breath method to measure liver glycogen oxidation.

Albumin synthesis in preterm infants on the first day of life studied with [1-13C]leucine.

Albumin is the major binding protein in the human neonate. Low production of albumin will lower its transport and binding capacity. This is especially important in preterm infants, in whom albumin binds to potentially toxic products such as bilirubin and antibiotics. To study the metabolism of plasma albumin in preterm infants, we administered a 24-h constant infusion of [1-(13)C]leucine to 24 very low birth weight (VLBW) infants (28.4 +/- 0.4 wk, 1,080 +/- 75 g) on the first day of life. The caloric intake consisted of glucose only, and therefore amino acids for albumin synthesis were derived from proteolysis. The fractional synthesis rate (FSR) of plasma albumin was 13.9 +/- 1.5%/day, and the absolute synthesis rate was 148 +/- 17 mg x kg(-1) x day(-1). Synthesis rates were significantly lower (P<0.03) in infants showing intrauterine growth retardation. Albumin synthesis increased with increasing SD scores for gestation and weight (P<0.05). The FSR of albumin tended to increase by 37% after administration of antenatal corticosteroids to improve postnatal lung function (P=0.09). We conclude that liver synthetic capacity is well developed in VLBW infants and that prenatal corticosteroids tend to increase albumin synthesis. Decreased weight gain rates in utero have effects on protein synthesis postnatally.

Albumin and whole-body protein synthesis respond differently to intraperitoneal and oral amino acids.

Patients with peritoneal dialysis are at risk for malnutrition and hypoalbuminemia, which are indicators of poor outcome. Recently, it was shown that dialysis solutions containing amino acids (AAs) and glucose improve protein anabolism in peritoneal dialysis patients. We determined if the same solutions could increase the fractional synthesis rate of albumin along with whole-body protein synthesis. Changes in the fractional albumin synthetic rate reflect acute change in hepatic albumin synthesis. A random-order cross-over study compared the effects of Nutrineal (AA source) plus Physioneal (glucose) dialysate with Physioneal alone dialysate. Eight patients in the overnight fasting state were compared to 12 patients in the daytime-fed state. Fractional albumin synthetic rate and whole-body protein synthesis were determined simultaneously using a primed-continuous infusion of L-[1-(13)C]-leucine. Fractional albumin synthesis on AAs plus glucose dialysis did not differ significantly from that on glucose alone in the fasting or the fed state. Protein intake by itself (fed versus fasting) failed to induce a significant increase in the fractional synthetic rate of albumin. Conversely, the oral protein brought about a significant stimulation of whole-body protein synthesis. Our findings show that the supply of AAs has different effects on whole-body protein synthesis and the fractional synthetic rate of albumin.

Immediate commencement of amino acid supplementation in preterm infants: effect on serum amino acid concentrations and protein kinetics on the first day of life.

To determine whether the general reluctance to begin amino acid administration to preterm infants from birth onward might lead to loss of lean body mass and impairment of growth, we measured amino acid levels and protein kinetics in 18 preterm infants. Nine infants received amino acids (1.15 +/- 0.06 gm.kg-1.day-1) and glucose (6.05 +/- 1.58 gm.kg-1.day-1), whereas the other nine infants received only glucose (6.48 +/- 1.30 gm.kg-1.day-1) from birth onward. Protein kinetics on the first postnatal day were measured with a stable isotope dilution technique with [1-13C]leucine as a tracer. No statistically significant differences were noted in blood pH, base excess, urea concentration, or glucose levels. Both total amino acid concentration and total essential amino acid concentration were significantly lower and were below the reference range in the nonsupplemented group. Plasma amino acid levels of five essential amino acids (methionine, cystine, isoleucine, leucine, arginine) were below the reference range in the nonsupplemented group, whereas only cystine was below the reference range in the supplemented group. Nitrogen retention was improved significantly by the administration of amino acids (-110 +/- 44 mg nitrogen per kilogram per day in the glucose-only group vs +10 +/- 127 mg nitrogen per kilogram per day in the group given glucose and amino acids; p = 0.001); leucine oxidation was not significantly increased in the supplemented group (41 +/- 13 mumol.kg-1.hr-1 vs 46 +/- 16 mumol.kg-1.hr-1). Leucine balance also improved significantly (-41 +/- 13 mumol.kg-1.hr-1 vs -8 +/- 16 mumol.kg-1.hr-1; p = 0.01) because of a combination of an increased amount of leucine being used for protein synthesis and a lower amount of leucine coming from protein breakdown. Plasma cystine concentration, the only amino acid below the reference range in the supplemented group, was highly predictive for protein synthesis in that group. We conclude that the administration of amino acids to preterm infants from birth onward seems safe and prevents the loss of protein mass.

Comparison of two preterm formulas with or without addition of medium-chain triglycerides (MCTs). I: Effects on nitrogen and fat balance and body composition changes.

Medium-chain triglycerides (MCTs) are included in the fat blend of several preterm formulas because of their complete absorption and rapid oxidation. The effects of two different fat blend compositions on nitrogen and fat balances and macronutrient oxidation were investigated in 28 healthy very-low-birth weight infants at 4 weeks of age. A preterm formula with a traditional corn oil/MCT blend containing 38% MCTs (MCT group) was compared to a new fat blend, designed to resemble human milk more, containing 6% MCTs (LCT group). There were no differences in nitrogen absorption or in excretion. Median nitrogen retention was 74% (MCT) vs. 71% (LCT) of intake. Fat absorption was higher (p less than 0.05) in the MCT group (88%) vs. 79% in the LCT group (median values). MCTs did not stimulate fat oxidation as measured by indirect calorimetry, so fat deposition was also higher on the MCT formula. As weight gain was not different between groups, the percentage of weight gain consisting of fat accretion was significantly (p less than 0.005) higher with the MCT formula (24% vs. 21%). On the other hand, there was no increase in percent protein accretion (both 15% of weight gain). We conclude that the existence of a slightly lower fat absorption in the healthy growing neonate fed a LCT formula compared with a MCT formula does not impair growth or nitrogen retention, but merely induces a slight decrease in the high relative fat accretion encountered in the preterm neonate.

Nocturnal oral glucose supplementation. The effects on protein metabolism in cirrhotic patients and in healthy controls.

Nocturnal glucose administration might prevent gluconeogenesis and concomitant protein loss due to hepatic glycogen depletion. In this study the effects of nocturnal oral glucose supplements on nitrogen metabolism were investigated in 8 cirrhotic patients and in 8 healthy controls. During the night, either polymeric glucose was given or water as placebo. In the patients with cirrhosis on placebo, nitrogen balance was not different from controls: -63 +/- 8 vs. -55 +/- 4 mg N/kg b.wt./9 h (mean +/- SEM). Cirrhotic patients had increased nocturnal protein turnover rates (measured with 15N-glycine) and increased early morning levels of free fatty acids (FFA), lactate, insulin, glucagon and growth hormone. After glucose, nitrogen balance improved by 36% in the cirrhotic group, with a decrease in protein turnover rates and a decrease in plasma levels of beta-hydroxybutyrate, urea and glucagon. In the controls, glucose had no effects on nitrogen balance, on protein turnover or on the hormone levels, except for reduced FFA and ketone body levels. These data show that nocturnal calorie supplements improve nitrogen balance during the night in cirrhotic patients but not in healthy controls. Long interprandial intervals should be avoided in cirrhotic patients.

Effects of early amino acid administration during total parenteral nutrition on protein metabolism in pre-term infants.

1. We investigated the effects of starting amino acid administration on post-natal day 2 on protein turnover and nitrogen balance in appropriate-for-gestational-age, very-low-birth-weight infants. Eighteen infants were divided into two groups. Group A received from day 2 onwards an amino acid solution, whereas group B started on this solution after day 4. Both groups were exclusively parenterally fed, 200 kJ day-1 kg-1 on post-natal days 3 and 4. Group A (birth weight 1.5 +/- 0.3 kg) received 4.6 g of glucose, 1.9 g of fat and 2.3 g of amino acids day-1 kg-1 body weight. Group B (birth weight 1.4 +/- 0.2 kg) received 7.0 g of glucose and 1.9 g of fat day-1 kg-1 body weight. 2. At post-natal day 3, a primed constant infusion of 3 mg of [15N]glycine day-1 kg-1 was given. Protein flux, protein synthesis and protein breakdown were calculated from the 15N enrichment in urinary ammonia. In five out of nine infants in group B no plateau of 15N enrichment in urinary urea could be detected, whereas in group A two out of nine infants did not reach a plateau. For this reason we did not use the end product urea for our calculations. 3. The administration of the amino acids resulted in a higher protein flux (6.9 +/- 1.5 g day-1 kg-1 versus 5.2 +/- 0.9 g day-1 kg-1) and a higher protein synthesis rate (6.0 +/- 1.4 g day-1 kg-1 versus 4.6 +/- 0.8 g day-1 kg-1) in group A.(ABSTRACT TRUNCATED AT 250 WORDS)

Muscle glycogen does not interfere with a 13CO2 breath test to monitor liver glycogen oxidation.

Naturally 13C-enriched carbohydrate has been used to label the liver glycogen pool for metabolic studies. The utilization of this glycogen was then monitored by the appearance of 13CO2 in breath. Using this method, it is assumed that during sedentary fasting the contribution of muscle glycogen towards oxidation is negligible. We investigated the influence of a different level of 13C enrichment of muscle glycogen on the 13C enrichment of breath CO2 while the breath test was carried out. In six healthy volunteers, the muscle glycogen stores were grossly depleted by a cycling exercise prior to consumption of the 13C-enriched diet which was given over a 10 h period. The oxidation of liver glycogen was measured during an 18 h sedentary fast. The results were compared with a control group who had not depleted their muscle glycogen before labelling. A higher 13C enrichment of muscle glycogen did not interfere with two parameters of liver glycogen oxidation, i.e. the duration of the plateau phase of 13CO2 and the return to baseline time. It was also shown that the 13C-labelled muscle glycogen was still available after the 18 h fast because a strenuous exercise led to a rapid 13CO2 enrichment. It is concluded that muscle glycogen 13C enrichment does not invalidate a 13CO2 breath test to measure liver glycogen oxidation during a sedentary fast.

Elevated protein requirements in cirrhosis of the liver investigated by whole body protein turnover studies.

1. In patients with cirrhosis of the liver and in healthy control subjects, the rates of nitrogen flux, protein synthesis and protein breakdown were studied, using a single oral dose of 200 mg of [15N]glycine as a tracer. The nitrogen flux through the amino acid pool was measured separately with both urinary ammonia and urinary urea as end products; the average value was used for further calculations. 2. Subjects were studied in the fed state, both on an adequate and a protein-restricted diet, and also in the fasting state. 3. The rates of protein synthesis were markedly increased in the patients, not only in the fed but also in the fasting state. Protein breakdown rates were increased in the patients in the fed state. 4. The nitrogen balance in steady-state conditions in the fed state was more positive in the patients, while their nitrogen loss in the fasting state was no higher than that of control subjects. 5. A hypothesis is put forward that the high protein requirements of cirrhotic patients could be caused by small and inadequate liver glycogen stores; due to these small stores, gluconeogenesis from amino acids will take place and lead to an extra amino acid loss even during short-term fasting. This increased amino acid loss could explain the elevated protein requirements in cirrhotic patients.

Evaluation studies of the 13C-mixed triglyceride breath test in healthy controls and adult cystic fibrosis patients with exocrine pancreatic insufficiency.

The 13C-mixed triglyceride (13C-MTG) breath test (BT) is a safe and noninvasive method to measure exocrine pancreatic function. We examined the reproducibility of the 13C-MTG BT in a group of 17 healthy controls and 8 adult patients with cystic fibrosis (CF). In controls no statistically significant difference in percentage dose recovered (PDR) was found between the first and the second result of repeated tests: the mean values were 35.5 +/- 5.5 vs. 32.3 +/- 7.4 PDR (n = 17). Also in the group of CF patients (n = 8) no significant difference between duplicate tests was found: mean values 17.5 +/- 7.5 and 17.5 +/- 7.8 PDR, respectively. The coefficient of repeatability is 8 PDR for the controls and CF patients together. Two factors might influence the outcome of the test. First, individually measured CO2 excretion instead of the usually assumed 9 mmol/h/kg CO2 production might alter the result of the 13CO2-MTG BT. Therefore CO2 production was measured by indirect calorimetry in 12 healthy controls and 13 CF patients. Measured CO2 excretion was not significantly different between healthy controls and CF patients. Secondly, exercise might influence BT results due to its separate effects on both CO2 production and excretion. The influence of physical exercise at a level of 25 or 50 W was studied on a bicycle ergometer in 4 healthy controls during the last 5 min of each 30-min sampling period. Exercise gave lower test results, on average 85% of the PDR value at rest. Incidently, it was observed in 1 patient that use of 13C-enriched food during the day preceding the test caused inappropriately low test results in the 13C-MTG BT. The 13C-MTG BT is a test with a fair but less than desirable reproducibility. Test conditions should be standardized to eliminate confounding influences. Exercise should be limited or strictly defined. Diet on the day preceding the test should not contain naturally 13C-enriched food. There is no need to measure individual CO2 production.

Whole-body protein turnover in preterm appropriate for gestational age and small for gestational age infants: comparison of [15N]glycine and [1-(13)C]leucine administered simultaneously.

Measurements of whole-body protein turnover in preterm infants have been made using different stable isotope methods. Large variation in results has been found, which could be due to different clinical conditions and/or the use of different tracers. We studied 14 appropriate for gestational age and nine small for gestational age orally fed preterm infants using [15N]glycine and [1-(13)C]leucine simultaneously, which allowed us to make a comparison of commonly used methods to calculate whole-body protein turnover. Whole-body protein turnover was calculated from 15N enrichment in urinary ammonia and urea after [15N]-glycine administration and from the 13C enrichment in expired CO2 after administration of [1-(13)C]leucine. Enrichment of alpha-ketoisocaproic acid after [1-(13)C]leucine constant infusion was measured as a direct parameter of whole-body protein turnover. Group means for whole-body protein turnover using [15N]glycine or [1-(13)C]leucine ranged from 10 to 14 g.kg-1.d-1, except when using the end product method that assumes a correlation between leucine oxidation and total nitrogen excretion. We found very low 15N enrichment of urinary urea in the majority of small for gestational age infants. These infants also had a lower nitrogen excretion in urine and oxidized less leucine. Nitrogen balance was higher in small for gestational age infants (416 +/- 25 mg.kg-1.d-1) compared with appropriate for gestational age infants (374 +/- 41 mg.kg-1.d-1, p = 0.003). [15N]Glycine does not seem to exchange its label with the body nitrogen pool to a significant degree and is therefore not always suitable as a carrier for 15N in protein turnover studies in premature infants.