Research goals for folate and related B vitamin in Europe.

In the past decade, the understanding of folate bioavailability, metabolism and related health issues has increased, but several problems remain, including the difficulty of delivering the available knowledge to the populations at risk. Owing to the low compliance of taking folic acid supplements, for example, among women of child-bearing age who could lower the risk of having a baby with a neural tube defect, food-based strategies aimed at increasing the intake of folate and other B-group vitamins should be a priority for future research. These should include the development of a combined strategy of supplemental folate (possibly with vitamin B(12)), biofortification using engineered plant-derived foods and micro-organisms and food fortification for increasing folate intakes in the general population. Currently, the most effective population-based strategy to reduce NTDs remains folic acid fortification. However, the possible adverse effect of high intakes of folic acid on neurologic functioning among elderly persons with vitamin B(12) deficiency needs urgent investigation. The results of ongoing randomized controlled studies aimed at reducing the prevalence of hyperhomocysteinemia and related morbidity must be available before food-based total population approaches for treatment of hyperhomocysteinemia can be recommended. Further research is required on quantitative assessment of folate intake and bioavailability, along with a more thorough understanding of physiological, biochemical and genetic processes involved in folate absorption and metabolism.

[6S]5-methyltetrahydrofolate or folic acid supplementation and absorption and initial elimination of folate in young and middle-aged adults.

OBJECTIVES: To assess the effects of supplementation with the diastereoisomer of 5-methyltetrahydrofolate ([6S]5-methylTHF), as an alternative supplement for folic acid, on folate absorption and elimination, in two age groups. DESIGN: A randomized, double-blind intervention study. SUBJECTS: A total of 12 young (<30 y) and 12 middle-aged (> or =50 y) healthy volunteers were recruited. METHODS: Volunteers were randomized to receive daily supplementation with 400 mug folic acid or equimolar amounts of [6S]5-methylTHF during 5 weeks. Before and after supplementation, absorption and initial elimination were calculated following oral [(2)H(2)]folic acid test doses using isotope kinetics in plasma. RESULTS: Folic acid absorption was lower in the middle-aged as compared to the young adults, both before (P = 0.03) and after (P = 0.05) supplementation. In the young adults, absorption decreased by 22% after [6S]5-methylTHF and increased by 21% after folic acid (P = 0.02). In the other age group, no such changes were found. The folate rate constant of elimination increased after folic acid supplementation in the young (+50%; P = 0.05) but not in the middle-aged (+18%; P = 0.5) adults. CONCLUSIONS: Young adults show increased folate turnover after folic acid supplementation relative to the effect of [6S]5-methylTHF supplementation. Similar differences are not observed in middle-aged adults, in whom folic acid absorption was found to be lower as compared to the young adults. SPONSORSHIP: Financial support was received from the European Union 5th Framework Programme (Grant QLRT-1999-00576).

Increasing fat in the diet does not improve muscle performance in patients with mitochondrial myopathy due to complex I deficiency.

Four myopathic patients with complex I deficiency followed diets containing 55 energy per cent (En%) as fat or 25 En% as fat, both for three weeks. Maximal workload and muscle force were not different on either diet. Exercise endurance time, oxygen consumption and lactate levels were also not different, but one patient had diminished endurance time on 25 En% as fat. Our observations do not support the use of increasing the fat in the diet of patients with mitochondrial complex I deficiency.

Oral estradiol decreases plasma homocysteine, vitamin B6, and albumin in postmenopausal women but does not change the whole-body homocysteine remethylation and transmethylation flux.

Estrogens, both endogenous and exogenous, lower the fasting levels of the independent risk factor for cardiovascular disease homocysteine. The mechanism behind this observation remains unclear. In a randomized, placebo-controlled, double-blind study, 25 postmenopausal women with a screening homocysteine concentration above 10 micromol/liter were included. We investigated the influence on homocysteine levels of a 3-month treatment with a daily oral dose of 4 mg 17beta-estradiol (ET) or 4 mg ET combined with 10 mg dydrogesterone (EPT); the comparison group received placebo treatment. We performed primed continuous infusions of L-[2H3-methyl-13C]methionine to assess steady-state flux rates of transmethylation, remethylation, and transsulfuration. Homocysteine concentration relationships with S-adenosylmethionine, S-adenosylhomocysteine, creatinine, albumin, vitamins B6 and B12, and folate status were determined as well. The mean change from baseline in homocysteine concentration by both treatments compared with placebo (ET, -13%; EPT, -10%) was accompanied by a decrease in the concentration of vitamin B6 (ET, -25%; EPT, -38%) and albumin (ET, -7%; EPT, -11%). No significant changes in flux rates were observed. In a .multiltivariate analysis, changes in homocysteine concentration were related to changes in albumin concentration. No relation to other variables was observed. We conclude that the ET- and EPT-induced homocysteine changes in this study were not accompanied by a significant change in methionine-homocysteine flux rates and hypothesize that an estrogen-induced lowering of homocysteine levels is primarily part of a change in albumin metabolism.

Hormone replacement influences homocysteine levels in the methionine-loading test: a randomized placebo controlled trial in postmenopausal women.

OBJECTIVE: To investigate the mechanism by which exogenous oestrogen influences the homocysteine metabolism in postmenopausal women. STUDY DESIGN: A randomized placebo controlled trial in which a methionine-loading test was performed, in 25 healthy postmenopausal women, before and after a 12-week oral treatment with placebo or daily 4 mg 17beta-estradiol with (HRT) or without (ERT) 10 mg dydrogesterone. Fasting and post-load homocysteine as well as Vitamins B(6), B(12) and folate were determined. RESULTS: In both treatment groups a significant 12% decrease in fasting homocysteine was observed. This decrease was accompanied by a post-load homocysteine increase of more than 20%. Vitamin B(6) values were decreased by more than 25%. CONCLUSION: The hormone therapy induced lowering of fasting homocysteine and Vitamin B(6) levels and an increase in post-load homocysteine, supporting the hypothesis that homocysteine-methionine metabolism is modulated by hormone therapy in postmenopausal women.

Nutritional support in 111 pediatric intensive care units: a European survey.

OBJECTIVE: To study current strategies in nutritional management of pediatric intensive care units (PICUs) in Europe, focusing on energy requirements. DESIGN AND SETTING: Survey by a 35-item questionnaire sent to representatives of 242 PICUs in 28 countries. Addresses were obtained from national PICU associations and the members' list of the European Society of Pediatric and Neonatal Intensive Care. PARTICIPANTS: Staff members of 111 European PICUs (46%) from 24 countries. MEASUREMENTS AND RESULTS: Predominantly physicians were reported to be responsible for nutritional support. In 73% of PICUs a multidisciplinary nutritional team was available. In most PICUs daily energy requirements were estimated using weight, age, predictive equations and correction factors. In 17% of PICUs energy expenditure was regularly measured by indirect calorimetry. Nutritional status was mostly assessed by weight, physical examination, and a wide range of biochemical blood parameters. Approximately 70% of PICUs used dedicated software for nutritional support. A similar percentage of PICUs regarded "nutrition" as a research topic and part of the residents' training program. CONCLUSIONS: Most European PICUs regard nutritional support as an important aspect of patient care, as shown by the presence of nutritional teams, software, research, and education. However, energy requirements of pediatric intensive care patient were based predominantly on estimations rather than on measurements.

5-Methyltetrahydrofolic acid and folic acid measured in plasma with liquid chromatography tandem mass spectrometry: applications to folate absorption and metabolism.

We describe a liquid chromatography (LC) tandem mass spectrometry (MS-MS) method for the determination of 5-methyltetrahydrofolic acid (5-methylTHF) and folic acid concentrations and enrichments in human plasma. It was used to study absorption and initial metabolism in five volunteers with two simultaneously administered oral test doses ([(13)C(6)]folic acid in capsules and [(2)H(2)]folic acid in a drink). [(13)C(5)]5-methylTHF and [(2)H(4)]folic acid were used as internal standards. Plasma samples (2 ml) were purified using folate binding protein affinity columns, followed by a concentration step. After LC separation, folates were detected using positive electrospray ionization MS-MS under multiple reaction monitoring conditions. Calibrations were linear for 5-methylTHF over the range 1.2 x 10(-11) (=limit of detection) to 3.2 x 10(-7)mol/L and for folic acid over the range 5 x 10(-10) (=limit of detection) to 4.5 x 10(-8)mol/L. For 5-methylTHF concentration in plasma, intraassay coefficient of variation was within 8.6% (and for unlabeled 5-methylTHF it was within 2.8%) and interassay coefficient of variation was within 9.0%. For folic acid concentrations these coefficient of variations were within 7.5% and within 6.5%, respectively. The [(13)C(6)] and [(2)H(2)] isotopomers of folic acid and 5-methylTHF were measured in the plasma of each volunteer for 8h. After accounting for the time delay due to capsule opening, the modeling results showed no significant differences in absorption time, first pass effect, and elimination rate in the folic acid test doses in capsule or drink. We conclude that LC-MS-MS offers increased sensitivity for quantification of plasma concentrations and enrichments of 5-methylTHF and folic acid and is applicable to stable-isotope studies in humans.

Short-term glucocorticoid treatment of piglets causes changes in growth plate morphology and angiogenesis.

OBJECTIVE: Glucocorticoid treatment of children often leads to growth retardation, and the precise target(s) in the growth plate responsible for this effect are unknown. Angiogenesis is an important part of the endochondral ossification process, and VEGF expressed in the growth plate is essential for proper angiogenesis to occur. Since glucocorticoid treatment down-regulates VEGF expression in cultured chondrocytes, we hypothesized that in vivo glucocorticoid treatment could result in VEGF down-regulation in the growth plate and disturbed angiogenesis, thus contributing to the growth retardation. DESIGN: We treated 6-week-old prepubertal piglets (10 kg) for 5 days with prednisolone (50 mg/day). Tibial growth plate sections were studied for apoptosis and the expression of VEGF protein and mRNA and MMP-9 protein. Capillaries in the metaphysis were visualized by CD31 immunostaining. Growth plate morphology (width of various zones) was determined by interactive measurements on hematoxylin/eosin stained sections and apoptotic cells were detected by TUNEL assay. RESULTS: In the prednisolone-treated animals, the total width of the growth plate decreased to 81% of controls (P<0.02), which was explained by a decrease of the width of the proliferative zone to 73% (P<0.05). The treatment had no effect on the orderly organization of the chondrocyte columns. In the growth plates of control animals, apoptosis was shown in 5.8% of the hypertrophic chondrocytes and was limited to the terminal hypertrophic chondrocytes. In prednisolone-treated animals, 40.5% of the hypertrophic chondrocytes was apoptotic (P<0.02), with apoptotic chondrocytes also appearing higher in the hypertrophic zone. We observed fewer capillaries and loss of their parallel organization in the metaphysis in the prednisolone-treated animals. The capillaries were shorter and chaotic in appearance. In contrast to controls, in prednisolone-treated animals VEGF mRNA and protein could not be detected in the hypertrophic zone of the growth plate. Trabecular bone length in the primary spongiosa was also diminished by the treatment. No changes were observed in the expression pattern of MMP-9, a matrix metalloproteinase, which is also important for angiogenesis and bone formation. CONCLUSIONS: These results indicate that short-term glucocorticoid treatment of growing piglets severely disturbs the width of the growth plate, apoptosis of chondrocytes, VEGF expression by hypertrophic chondrocytes, the normal invasion of blood vessels from the metaphysis to the growth plate and bone formation at the chondro-osseous junction. These effects could alter the dynamics of endochondral ossification and thus contribute to glucocorticoid-induced growth retardation.

Energy expenditure in infants with congenital heart disease, including a meta-analysis.

AIM: To assess energy requirements and body composition in preoperative children with congenital heart disease (CHD). METHODS: In 11 infants with CHD (2-8 mo), total daily energy expenditure (TDEE) and total body water (TBW) were measured with doubly labelled water and compared with historic data from healthy controls. Within the patient group, energy expenditure of infants with versus those without congestive heart failure was compared. Subsequently, the data were pooled with literature data in meta-analyses. RESULTS: CHD patients showed increased TBW (mean +/- SD 66 +/- 3 vs 58 +/- 5% of body weight, p < 0.05) and energy expenditure (381 +/- 42 vs 298 +/- 36 kJ kg(-1) d(-1), p < 0.001). Meta-analyses showed that CHD infants have 35% increased TDEE (376 vs 278 kJ kg(-1) d(-1) , p < 0.00001) and 7% higher TBW (p < 0.0001). Coexistent congestive heart failure (treated with diuretics) had no influence on TDEE (mean difference 14 kJ kg(-1) d(-1) , not significant). In patients with heart failure and growth retardation, an energy balance study showed an average 12% loss of initially ingested energy due to vomiting, increased TDEE and low faecal energy loss, resulting in low energy available for growth, compared with controls (42 +/- 30 vs 96 +/- 61 kJ kg(-1) d(-1) , p < 0.05). CONCLUSION: Many infants with CHD require substantially higher energy intake (at least 100 kJ kg(-1) d(-1) extra) owing to increased TDEE, which is not explained by a higher percentage of body water. Coexistent heart failure does not appear to have an additional influence on TDEE. In infants with CHD and growth failure factors other than elevated TDEE, including vomiting, may explain the disturbed energy balance.

Determination of 13C and 15N enrichments of urea in plasma by gas chromatography-combustion isotope ratio mass spectrometry and gas chromatography-mass spectrometry using the 2-methoxypyrimidine derivative.

We describe a GC-MS and GC-c-IRMS method for the determination of labeled urea tracer enrichments in plasma as a result of combined 13C- and 15N(2)-urea infusion experiments in piglets. Urea was converted into 2-methoxypyrimidine, a stable derivative, suited for analyses by both GC-MS and GC-c-IRMS. Using calibration curves for the respective working ranges (13C-urea: 0-1% APE; 15N(2)-urea: 0-7% MPE) enrichments were established in single point measurements; for 15N(2)-urea as values+/-0.15% MPE (95% confidence interval); for 13C-urea as values+/-0.02% APE (95% confidence interval). 15N(1)-urea enrichments were determined by measurement of the same sample with GC-c-IRMS and GC-MS. Subtraction of the 13C specific GC-c-IRMS data from the nondiscriminating GC-MS data for the sum of 13C- and 15N(1)-urea resulted in 15N(1)-urea enrichments+/-0.15% MPE (95% confidence interval). Application of the method in a combined 13C-urea bolus and 15N(2)-urea primed constant infusion experiment in piglet was demonstrated.

S-adenosylhomocysteine and the ratio of S-adenosylmethionine to S-adenosylhomocysteine are not related to folate, cobalamin and vitamin B6 concentrations.

BACKGROUND: It is unclear whether homocysteine itself is causal in the pathogenesis of cardiovascular disease. Alternatively or additionally, the association between homocysteine and cardiovascular disease may be because of its metabolic precursor, S-adenosylhomocysteine, or of the ratio of S-adenosylmethionine to S-adenosylhomocysteine. Therefore, it is relevant to know how these moieties are interrelated, and whether, as is the case for homocysteine, they are influenced by blood levels of folate, cobalamin or vitamin B6. DESIGN: We cross-sectionally studied a population-based cohort of 97 Caucasian subjects aged 60-85 years. Concentrations of homocysteine, S-adenosylhomocysteine, S-adenosylmethionine, folate, cobalamin and vitamin B6 were measured in fasting blood samples. RESULTS: In multiple regression analysis, homocysteine was associated with vitamin B12 (per 50 pmol L-1 increase of cobalamin, change in homocysteine, -0.70 mmol L-1; 95% CI, -1.30 to -0.10 mmol L-1) and folate (per 100 nmol L-1 increase in erythrocyte folate, change in homocysteine, -0.68 mmol L-1; 95% CI -1.28 to -0.08 mmol L-1). S-adenosylhomocysteine, S-adenosylmethionine and the ratio of S-adenosylmethionine to S-adenosylhomocysteine were not associated with serum folate, cobalamin or vitamin B6, nor with erythrocyte folate. Furthermore, plasma homocysteine showed a negative correlation with the ratio of S-adenosylmethionine to S-adenosylhomocysteine in plasma (r = -0.27; P < 0.01) but not in erythrocytes. CONCLUSIONS: In contrast to homocysteine, the plasma concentrations of S-adenosylhomocysteine and the ratio of S-adenosylmethionine to S-adenosylhomocysteine were not associated with the folate, cobalamin and vitamin B6 concentrations in the present study. If these precursors in part explain why homocysteine is associated with cardiovascular disease, homocysteine-lowering treatment with B vitamins may be less effective than currently expected, at least in an elderly population.

Resting oxygen consumption and in vivo ADP are increased in myopathy due to complex I deficiency.

BACKGROUND: Patients with isolated complex I deficiency (CID) in skeletal muscle mitochondria often present with exercise intolerance as their major clinical symptom. OBJECTIVE: To study the in vivo bioenergetics in patients with complex I deficiency in skeletal muscle mitochondria. METHODS: In vivo bioenergetics were studied in three of these patients by measuring oxygen uptake at rest and during maximal exercise, together with forearm ADP concentrations ([ADP]) at rest. Whole-body oxygen consumption at rest (VO(2)) was measured with respiratory calorimetry. Maximal oxygen uptake (VO(2)max) was measured during maximal exercise on a cycle ergometer. Resting [ADP] was estimated from in vivo (31)P MRS measurements of inorganic phosphate, phosphocreatine, and ATP content of forearm muscle. RESULTS: Resting VO(2) was significantly increased in all three patients: 128 +/- 14% (SD) of values in healthy control subjects. VO(2)max in patients was on average 2.8 times their VO(2) at rest and was only 28% of VO(2)max in control subjects. Resting [ADP] in forearm muscle was significantly increased compared with healthy control subjects (patients 26 +/- 2 microM, healthy controls 9 +/- 2 microM). CONCLUSION: In patients with CID, the increased whole-body oxygen consumption rate at rest reflects increased electron transport through the respiratory chain, driven by a decreased phosphorylation potential. The increased electron transport rate may compensate for the decreased efficiency of oxidative phosphorylation (phosphorylation potential).

[Karl Landsteiner (1868-1943) and the specificity of serological reactions, one hundred years ago and now]. / Karl Landsteiner (1868-1943) en de specificiteit van serologische reacties, honderd jaar geleden en nu.

One hundred years after his discovery of the AB0 blood groups, the monumental work of Austrian physician Karl Landsteiner (1868-1943, Nobel Prize 1930) on the specificity of serological reactions is widely recognised. His work on isoantigen reactions included the discovery, together with Levine and Wiener, of the Rhesus factor. Landsteiner did much to develop our understanding of autoantibody reactions and also contributed insights into the aetiology of infectious diseases that were rampant in his time. His work in immunochemistry included the discovery of hapten antigenicity. However, most of all he wanted to be remembered for his theory on the specificity of serological reactions, which he had strongly substantiated with his own work. Landsteiner suffered from the shortages in Vienna at the end of World War I. He and his family moved to the Netherlands in 1919. There he worked as pathologist at the Red Cross Hospital in The Hague until his move to the Rockefeller Institute in New York in 1923. Nowadays, his work is celebrated for its numerous excellent contributions to the fields of transfusion and transplantation medicine, population genetics, immunology, and infectiology.

Insulin-like growth factors and leucine kinetics during exercise training in children with cystic fibrosis.

BACKGROUND: Little is known about the metabolic effects of exercise training in children with cystic fibrosis. The hypothesis for the current study was that in patients with declining clinical status, exercise increases circulating insulin-like growth factors (IGFs) and improves protein kinetics. METHODS: This was a prospective intervention study in 10 children with cystic fibrosis who participated in a structured isoenergetic exercise (cycling) training program for 3 months. Measurements of IGFs, protein kinetics (using intravenous [13C]-1-leucine tracer infusions) and nutritional balance studies were conducted at baseline and after 3 months. RESULTS: Standard deviation scores of plasma IGF-I, IGF-II, and IGF binding protein (BP)-3 were all decreased at baseline (mean +/- SE: -2.0+/-0.2, -2.0+/-0.2. -0.6+/-0.2, respectively). IGF-I and IGF-II concentrations were significantly higher after exercise training (standard deviation scores -1.4+/-0.3 and -1.3 +/-0. 1, respectively; compared with baseline: one-tailed t-test P = 0.03 and 0.002). The standard deviation score of the IGF-I/IGF BP-3 ratio, an indicator of free IGF-I in the circulation, normalized during exercise training (0.0+/-0.6 vs. -1.3+/-0.2 SD units at baseline, one-tailed t-test P = 0.04). There was no significant difference in protein intake and fasting protein breakdown, oxidation, and protein synthesis or in energy balance and fat absorption. CONCLUSIONS: These results show that isoenergetic exercise training can be safely recommended to patients with cystic fibrosis. It provides a positive anabolic stimulus to IGF status but is not sufficient to adequately augment protein accretion in patients with diminished nutritional status.

Determination of S-adenosylmethionine and S-adenosylhomocysteine in plasma and cerebrospinal fluid by stable-isotope dilution tandem mass spectrometry.

BACKGROUND: Available methods for the determination of nanomolar concentrations of S:-adenosylmethionine (SAM) and S:-adenosylhomocysteine (SAH) in plasma and cerebrospinal fluid (CSF) are time-consuming. We wished to develop a method for their rapid and simultaneous measurement. METHODS: We used tandem mass spectrometry (MS/MS) for the simultaneous determination of SAM and SAH, with stable-isotope-labeled internal standards. The (13)C(5)-SAH internal standard was enzymatically prepared using SAH-hydrolase and [(13)C(5)]adenosine. The method comprises a weak anion-exchange solid-phase extraction procedure serving as clean-up step for the deproteinized plasma and CSF samples. After clean-up, samples were injected on a C(18) HPLC column, which was connected directly to the tandem mass spectrometer, operating in MS/MS mode. RESULTS: In plasma samples, the intraassay CVs for SAM and SAH were 4.2% and 4.0%, respectively, and the interassay CVs were 7.6% and 5. 9%, respectively. In CSF, the intraassay CVs for SAM and SAH were 6. 8% and 6.9%, respectively, and the interassay CVs were 4.2% and 5.5%, respectively. Mean recovery of SAM and SAH for both matrices at two concentrations was 93%. Detection limits for SAM and SAH in samples were 7.5 and 2.5 nmol/L, respectively. Concentrations of SAM and SAH in plasma from healthy subjects were within the previously reported ranges. In 10 CSF samples, the mean concentrations (range) were 248 (137-385) nmol/L for SAM and 11.3 (8.9-14.1) nmol/L for SAH. CONCLUSIONS: SAM and SAH can be analyzed by MS/MS, taking optimal advantage of the speed and high sensitivity and specificity of this relatively new analytical technique.

A single food bolus stimulates albumin synthesis in growing piglets.

BACKGROUND: A stable isotope tracer method to quantify the synthesis of proteins of hepatic origin in response to feeding is described. The response of albumin synthesis on one mixed meal in a piglet model was investigated and the intragastric and intravenous administration modes of 13C-valine were compared. METHODS: The fasting and postprandial fractional synthesis rates (FSRs) of albumin in 15 piglets were measured while infusion rates of 13C-valine were changed in anticipation of the increased appearance of the tracee after a single liquid food bolus (30 mL/kg infant formula). 13C-valine enrichments in albumin hydrolysates at regular time intervals were determined with gas chromatography-combustion isotope ratio mass spectrometry. RESULTS: The intravenous mode (n = 8) showed constant plasma alpha-ketoisovalerate tracer-to-tracee ratios (coefficient of variation range: 1-8%), and a 27% increase in albumin FSR after the food bolus (mean FSR +/- standard error [SE]: fasting 14.4% +/- 1.6% vs. postprandial 18.3% +/- 2.2% per day; P < 0.005). In the intragastric mode (n = 7), albumin FSR calculated from the mean precursor values increased 32% after feeding (fasting 14.6% +/- 1.5% vs. postprandial 19.3% +/- 1.6% per day; P = 0.005), despite absence of constant alpha-ketoisovalerate enrichment (coefficient of variation range: 15-31%). The FSRs were not significantly different between both infusion modes. CONCLUSIONS: A mixed food bolus increases albumin FSR in growing piglets by approximately 30%, irrespective of the tracer administration route. The concept of anticipated precursor steady state is applicable to study changes of hepatic protein synthesis after a single meal. The intragastric mode of tracer administration can be applied as a less invasive method to measure tissue specific protein synthesis in children.

Nephrotic proteinuria has No net effect on total body protein synthesis: measurements with (13)C valine.

In nephrotic syndrome, significant amounts of plasma proteins, mostly of hepatic origin, are lost in urine. Total hepatic protein synthesis increases, suggesting that other protein pools must be conserved to maintain steady state. This can be accomplished either by decreased amino acid oxidation or decreased protein synthesis in other organs to replace lost liver-derived proteins. To determine the effect of nephrotic syndrome on total-body protein metabolism, we compared whole-body valine use in seven nephrotic patients and five controls using a primed continuous infusion of [1-(13)C]-valine, with additional priming of NaH(13)CO(3). Plasma [(13)C]-valine, (13)C alpha ketoisovaleric acid, and the expired (13)CO(2) enrichments were used to assess whole-body valine flux, valine oxidation, and nonoxidative valine disposal (NOVD). The valine flux into the blood compartment (97.7 +/- 3.0 versus 95.3 +/- 3.3 micromol/kg/h), oxidation of valine (19.4 +/- 1.9 versus 21.2 +/- 2. 8 micromol/kg/h), and NOVD (78.3 +/- 2.5 versus 74.2 +/- 2.7 micromol/kg/h) were not statistically different in patients compared with controls. Valine oxidation correlated positively with urinary urea excretion (r = 0.70; P = 0.01) in all subjects. Compared with control subjects who have similar urinary urea excretion, nephrotic subjects do not compensate for urinary loss of protein by decreased amino acid oxidation or decreased nonoxidative valine disposal. Previous studies have shown that synthesis of several hepatic proteins increases when subjects are fed the same dietary regime, whereas the present study shows that total-body protein synthesis does not increase. This would imply reduced synthesis of nonhepatic protein pools.

Determination of isotopic enrichments of [1-13C]homocysteine, [1-13C]methionine and [2H3-methyl-1-13C]methionine in human plasma by gas chromatography-negative chemical ionization mass spectrometry.

We describe a reliable method for the simultaneous determination of isotopic enrichments of [1-13C]homocysteine, [1-13C]methionine and [2H3-methyl-1-13C]methionine in human plasma. Accurate [1-13C]homocysteine calibration standards were prepared by chemical conversion via thiolactonisation of [1-13C]methionine standards. Based upon anion-exchange chromatography, (di)acetyl-3,5-bis-trifluoromethylbenzyl derivatives, preparation of accurate calibration curves and gas chromatography-negative chemical ionization mass spectrometry, isotopic enrichments in human plasma could be determined with TTR (%) <+/-0.2% (N=3) for [1-13C]homocysteine (enrichment range 0-8%), [1-13C]methionine (enrichment range 0-3%) and [2H3-methyl-1-13C]methionine (enrichment range 0-12%). The method was applied in a [2H3-methyl-1-13C]methionine tracer infusion study in a biological model.