Meal replacement reduces insulin requirement, HbA1c and weight long-term in type 2 diabetes patients with >100 U insulin per day.

BACKGROUND: Despite high insulin doses, good glycaemic control is often lacking in type 2 diabetes patients and new therapeutic options are needed. METHODS: In a proof of principle study, an energy-restricted, protein-rich meal replacement (PRMR) was examined as a means of reducing insulin requirement, HbA1C and body weight. Obese type 2 diabetes patients (n = 22) with >100 U insulin per day replaced, in week 1, the three main meals with 50 g of PRMR (Almased-Vitalkost) each (= 4903 kJ day(-1) ). In weeks 2-4, breakfast and dinner were replaced, and, in weeks 5-12, only dinner was replaced. Clinical parameters were determined at baseline, and after 4, 8 and 12 weeks, as well as after 1.5 years of follow-up. The Wilcoxon signed-rank test was used for the intention-to-treat analysis and the Mann-Whitney U-test for subgroup analyses. RESULTS: The 12-week-programme was completed by 15 participants (68%). After 1 week, the mean insulin dose was reduced from 147 (75) U to 91 (55) U day(-1) (P = 0.0001), and to 65 (32) U (P < 0.0001) after 12 weeks of study. Over a period of 12 weeks, HbA1c decreased from 8.8% (1.4%) to 8.1% (1.6%) (P = 0.048) and weight decreased from 118.0 (19.7) kg to 107.4 (19.2) kg (P < 0.0001). Moreover, body mass index, waist and hip circumference, fasting blood glucose, triglycerides and high-density lipoprotein cholesterol improved significantly. After 1.5 years, insulin requirement and weight remained significantly lower than baseline. Participants who continued PRMR further reduced their HbA1c, weight and insulin dose. Two patients were able to stop insulin therapy altogether. CONCLUSIONS: Energy-restricted PRMR was effective in reducing insulin requirement of type 2 diabetes patients with intensified insulin therapy accompanied by a reduction of HbA1c, weight and other cardiometabolic risk factors. With the continuous use of PRMR, glycaemic control might be improved in the long term.

Description of the mutations in 15 subjects with variant forms of maple syrup urine disease.

BACKGROUND: In maple syrup urine disease (MSUD), disease-causing mutations can affect the BCKDHA, BCKDHB or DBT genes encoding for the E1 alpha, E1 beta and E2 subunits of the multienzyme branched-chain 2-keto acid dehydrogenase (BCKD) complex. AIM: The aim of this study was to screen DNA samples of 15 subjects with distinct well-characterized variant MSUD phenotypes for mutations in the three genes in order to demonstrate a potential correlation between specific nucleotide changes and particular variant phenotypes. METHODS: The exonic coding sequences of all three genes were studied using genomic DNA and cellular RNA derived from peripheral blood leukocytes. RESULTS: In 37% of the cases (total 30 alleles), disease-causing mutations were located in the BCKDHA, in 46% in the BCKDHB, and in 13% in the DBT gene. Novel mutations occurring homozygously were p.Ala328Thr in the BCKDHA gene and p.Gly249_Lys257del in the DBT gene. Both are associated with a mild MSUD variant. The same holds true for the novel mutations p.Pro200Ala in BCKDHB and p.Phe307Ser in DBT which were identified in heterozygous fashion. Among the known mutant alleles, p.Gly278Ser in the BCKDHB gene was relatively frequent and also associated with a mild MSUD variant. CONCLUSION: The results of this study indicate that genotyping may be predictive of clinical severity of variant MSUD phenotypes and might be of prognostic value particularly in subjects with variant MSUD identified in newborn screening in whom early treatment fortunately slows the natural course of the disease.

Variant maple syrup urine disease (MSUD)--the entire spectrum.

BACKGROUND: In the rare inborn autosomal recessive disorder maple syrup urine disease (MSUD) the accumulation of the branched-chain amino acids (BCAAs) and their metabolic products results in acute and chronic brain dysfunction. About 20% of the patients suffer from non-classic variant forms of MSUD of different clinical severity. AIM: Up to now variant cases have mostly been published as individual case reports; the aim of this study was to give a comparative description of 16 individuals (aged 6-30 years) with different forms of variant MSUD. METHODS: Laboratory data, information on clinical course and treatment as well as aspects of developmental, intellectual and social outcome were obtained retrospectively. Data from in vitro and in vivo methods measuring the degree of enzyme deficiency were included. RESULTS: In addition to a mild phenotype, which fits well into the so-called intermittent variant, and a more severe phenotype with a wider range from a mild variant to an almost classic form, which fits well into the so-called intermediate variant, we assume the existence of an asymptomatic, non-disease variant of MSUD. These clinical phenotypes are not unambiguously differentiable on the basis of biochemical parameters. CONCLUSION: A continuum of clinical severity from asymptomatic to very severe (border to classic) exists in variant MSUD. Apart from newborns with classic MSUD, also those with variant forms benefit from early diagnosis and start of adequate treatment.

Transamination and oxidative decarboxylation of L-isoleucine, L-alloisoleucine and related 2-oxo acids in perfused rat hind limb muscle.

Metabolism of L-isoleucine, L-alloisoleucine and corresponding 2-oxo acids in rat hind limb muscle was comparatively studied under steady-state perfusion conditions. At 0.5 mM L-[1-14C]isoleucine, apparent transamination and 2-oxo acid decarboxylation rates amounted to about 17 and 4 nmol/min per g of muscle, respectively. With L-allo[1-14C]isoleucine, the corresponding rates were about 5- and 10-fold lower, respectively. After addition of dichloroacetate (1-5 mM), the portion of (S)- and (R)-methyl-2-oxopentanoate undergoing further oxidative decarboxylation within the tissue was similarly increased by over 40%. In perfusions with 0.5 mM (R,S)-3-methyl-2-oxopentanoate and tracer doses of 1-14C-labeled (S)- or (R)-enantiomer, the 14CO2 production was comparable (about 0.5 nmol/min per g of muscle). Dichloroacetate caused a several-fold increase in 14CO2 release from either enantiomer, apparent 2-oxo acid transamination rates remaining unaffected. Indications for a racemization of 2-oxo acid were not obtained in the experiments. The results are discussed with respect to the appearance/disappearance of L-alloisoleucine in vivo and to the fact that (R)-3-methyl-2-oxopentanoate, but not L-alloisoleucine, can support growth of rats on a diet deficient in L-isoleucine.

Differential effect of dichloroacetate on branched-chain amino acid catabolism in perfused rat hindlimbs.

The effect of 1 mM and 5 mM dichloroacetate on the catabolism of branched-chain amino acids in isolated rat hindlimbs was investigated in perfusions with 0.5 mM 1-14C-labeled L-leucine or L-valine. The results demonstrate an increasing effect of dichloroacetate on the flux through skeletal muscle branched-chain 2-oxo acid dehydrogenase. A minor effect was observed with the high dichloroacetate concentration. Evidence is presented that this was essentially due to diminished pyruvate supply.

Effect of adrenergic agonists on phosphoinositide breakdown in rat skeletal muscle preparations.

Adrenergic regulation of phosphoinositide breakdown in rat skeletal muscle was investigated in 30-min incubations with 10 mM LiCl. In rat hemidiaphragms, prelabelled with D-myo-[2-3H]inositol, addition of alpha-agonists (epinephrine, norepinephrine, phenylephrine) induced a 5-8-fold increase of [3H]inositol monophosphate accumulation. This could be prevented by inclusion of alpha-antagonists (phentolamine, prazosin). beta-Agonists and/or beta-antagonists had no effect. Similar experiments with isolated flexor digitorum brevis muscle fibers yielded confirmatory results. Functional integrity of beta-receptor mediated processes was suggested by the beta-agonist-induced increase of glucose 6-phosphate in hemidiaphragms and cAMP in fiber preparations. The results indicate that phosphoinositide breakdown in differentiated rat skeletal muscle is, at least in part, under alpha-adrenergic control.

Analysis of maple syrup urine disease in cell culture: use of substrates.

Branched-chain 2-oxo acid dehydrogenase activity in human skin fibroblasts against L-leucine, L-valine, L-isoleucine and derived 2-oxo acids was compared in incubations with 1 mmol/l of 1-14C-labelled substrate. The results suggested that the amino acids are the more suitable substrates for an estimation of decarboxylation activity in intact cells. In control cell lines (n = 12), 14CO2 release from amino acids was highest for valine and least for leucine. In a representative number of fibroblast strains of patients with different forms of MSUD (n = 11; residual decarboxylation activity 2-60% of the controls), 14CO2 release from the different amino acids was reduced to a similar degree. Additional measurement of 2-oxo[1-14C]acid release suggested that substrate supply to the branched-chain 2-oxo acid dehydrogenase complex was not rate limiting in the cell lines under investigation.

A convenient enzymatic method for the determination of 4-methyl-2-oxopentanoate in plasma: comparison with high performance liquid chromatographic analysis.

A simple and rapid spectrophotometric method for the estimation of 4-methyl-2-oxopentanoate in plasma samples by use of NAD+-dependent D-2-hydroxyisocaproate dehydrogenase from Lactobacillus casei ssp. pseudoplantarum is described. It is based on the kinetic measurement of the decrease of NADH absorbance at 334 nm. Applicability is demonstrated by comparative measurement of 4-methyl-2-oxopentanoate content in plasma of patients with maple syrup urine disease by the enzymatic and a reversed phase high performance liquid chromatographic method.

Determination of (S)- and (R)-2-oxo-3-methylvaleric acid in plasma of patients with maple syrup urine disease.

An enzymatic method for the separate measurement of both chiral 2-oxo-3-methylvaleric acid (OMV) compounds, (S)- and (R)-OMV, by NADH-dependent enantioselective amination using leucine dehydrogenase in the presence of a NADH regenerating system is described. This method allows the quantitative determination of all branched-chain 2-oxo acids, simultaneously. In plasma samples from classical maple syrup urine disease patients under therapy the average (R)-OMV/(S)-OMV ratio was 0.35 and great differences in the transamination equilibria of the diastereomeric branched-chain amino acids L-isoleucine and L-alloisoleucine were demonstrated.

Significance of diagnostic parameters in [(13)c]octanoic Acid gastric emptying breath tests*.

Abstract Two novel characteristic parameters, the latency time (t (lat)) and the ascension time (t (asc)), are proposed for evaluation of non-invasive [(13)C]octanoic acid breath tests for assessment of the gastric emptying of solids. In breath tests performed in control subjects (n = 30) and diabetic patients (n = 100), the usefulness of these parameters was compared to conventional parameters, i.e., gastric half emptying-time t (1/2,b )) and lag phase (t (lag,b )). The proposed parameters were only loosely correlated (controls, r = 0.199; diabetics, 0.616). A strong correlation was found between the conventional parameters (controls, r = 0.891; diabetics, r = 0.962). Based on the conventional method, 36 patients were suspicious of delayed gastric emptying including 24 patients which exhibited a simultaneous delay in both parameters. Using the new parameters, a total of 46 patients were suspicious of delayed gastric emptying with 15 and 20 having isolated delay in t (lat) and t (asc), respectively. We conclude that the novel parameters may be more appropriate for examination of the different phases of gastric emptying and for evaluation of gastric emptying disturbances in diabetic patients than the parameters conventionally used for this purpose.

Significance of diagnostic parameters in [13C]octanoic acid gastric emptying breath tests.

Two novel characteristic parameters, the latency time (t(lat)) and the ascension time (t(asc)), are proposed for evaluation of non-invasive [13C]octanoic acid breath tests for assessment of the gastric emptying of solids. In breath tests performed in control subjects (n = 30) and diabetic patients (n = 100), the usefulness of these parameters was compared to conventional parameters, i.e., gastric half emptying-time (t1/2,b) and lag phase (t(lag),b). The proposed parameters were only loosely correlated (controls, r = 0.199; diabetics, 0.616). A strong correlation was found between the conventional parameters (controls, r = 0.891; diabetics, r = 0.962). Based on the conventional method, 36 patients were suspicious of delayed gastric emptying including 24 patients which exhibited a simultaneous delay in both parameters. Using the new parameters, a total of 46 patients were suspicious of delayed gastric emptying with 15 and 20 having isolated delay in t(lat) and t(asc), respectively. We conclude that the novel parameters may be more appropriate for examination of the different phases of gastric emptying and for evaluation of gastric emptying disturbances in diabetic patients than the parameters conventionally used for this purpose.

Compartmental approach for evaluation of plasma kinetics and (13)co(2)-exhalation after oral loading with L-[1-(13)c]leucine.

Abstract A seven compartment model was applied for evaluation of oral L-[1-(13)C]leucine loading tests (38 µmol/kg body wt.) in healthy volunteers. The model comprises transport and absorption in stomach and gut into a central L-leucine-compartment which is connected to a protein compartment and to the compartment of the corresponding 2-oxo acid. CO(2) release from the latter occurs in a fast and a slow compartment into the central CO(2) compartment for exhalation. Using the fmins routine of MATLAB for parameter estimation, a good agreement was obtained between calculated and actually measured kinetics of (13)C-labelled metabolites and a mean in vivo L-leucine oxidation of 0.365 ± 0.071 µmol/kg per min (n = 5) was computed. Plausibility of the model was checked by predicting in vivo leucine oxidation rates from primed continuous infusion tests (priming: L-[1-(13)C]leucine, 5 µmol/kg; NaH(13)CO(2), 1.2 µmol/kg; infusion: L-[1-(13)C]leucine, 5 µmol/kg per h). In 5 tested volunteers, the experimental L-leucine oxidation rate amounted to 0.358 ± 0.105 µmol/kg per min versus predicted 0.324±0.099 µmol/kg per min. Possible causes for some observed intraindividual variations are discussed.

[Is homocysteine a risk factor for coronary heart disease in patients with terminal renal failure?]. / Ist Homocystein ein Risikofaktor für das Vorliegen einer koronaren Herzkrankheit bei Patienten mit terminaler Niereninsuffizienz?

BACKGROUND: Cardiovascular disease is a major cause of mortality in chronic uremic patients. We studied whether homocysteine is an independent cardiovascular risk factor for patients with end-stage renal disease (ESRD). PATIENTS AND METHODS: The study included 163 patients and controls (Group 1: healthy controls, n = 20; Group 2: patients with chronic renal failure, serum creatinine < or = 4 mg/dl, n = 23; Group 3: patients with ESRD, n = 91; Group 4: renal transplant recipients, serum creatinine < or = 2.5 mg/dl, n = 29). We registered patients for the following factors: age, diabetes, smoking, lipids, vitamin B12, folic acid and homocysteine. The coronary heart disease was diagnosed by coronary angiography. RESULTS: The cardiovascular risk profile (hypertension, diabetes, smoking, hyperlipidemia) among uremic patients was significantly increased compared to the healthy controls. There was a significant correlation between the impairment of renal function and the increase of the homocysteine concentration (Group 1: 12 +/- 4.3 mumol/l vs Group 3: 27.8 +/- 15.8 mumol/l; p < 0.001). There was no significant difference of homocysteine between the patients with coronary heart disease and those without (29.9 +/- 18.1 mumol/l vs 26.6 +/- 14.4 mumol/l, not significant). CONCLUSION: In this study a significant correlation between the number of cardiovascular risk factors and the incidence of cardiovascular disease was proven. Although homocysteine was increased among patients with impaired renal function, hyperhomocysteinemia could not be identified as a significant risk factor for coronary heart disease in patients with ESRD. It is assumable that the pathogenesis of coronary heart disease in patients with ESRD is of multifactorial origin.

[The isolated perfused hindlimb of the rat. Method and use]. / Das isoliert perfundierte Hinterbein der Ratte. Methodik und Anwendung.

Diaphragm and isolated perfused hindquarter of rat are among the most thoroughly studied skeletal muscle preparations. Preparation and perfusion techniques are described. The preparation is characterized with respect to tissue and muscle fiber type composition as well as functional metabolic parameters. Some applications are demonstrated.

Comparison of the catabolism of branched-chain L-amino acids in cultured human skin fibroblasts.

Using 1-14C-labeled substrates, the metabolism of naturally occurring branched-chain L-amino acids was studied in incubations with cultured human skin fibroblasts derived from normal subjects and from a patient with maple syrup urine disease (variant form). Practically saturating conditions were reached at 1 mmol/liter of substrate and metabolic rates remained essentially constant up to 120 min. In control fibroblasts, the transamination of 14C-labeled leucine, valine, isoleucine, and allo-isoleucine (1 mmol/liter) was about 26, 13, 12, and 5 nmol/90 min/mg of cell protein, respectively. The portion of transamination products undergoing oxidative decarboxylation within the cells was about 17, 43, 34, and 23%, respectively. With the maple syrup urine disease cell line, comparable transamination rates were found. As compared to the findings with normal cells, however, 14CO2 production from the above mentioned substrates was reduced and amounted to 14, 11, 25, and 45%, respectively. Thus it appeared that residual branched-chain 2-oxo acid dehydrogenase activity was differently reduced towards the four 2-oxo acid substrates.

Functional differences in the catabolism of branched-chain L-amino acids in cultured normal and maple syrup urine disease fibroblasts.

Possible functional differences in the catabolism of the four branched-chain L-amino acids in maple syrup urine disease were assessed using cultured human skin fibroblast stains. Transamination and oxidative decarboxylation were comparatively studied in 90-min incubations with 1 mmole/liter of 1-14C-labeled substrates. In normal cell strains (n = 5), apparent transamination rates (sum of branched-chain 2-oxo[14C]acid and 14CO2 release; means expressed in nmole/90 min/mg of cell protein) were in the order L-leucine (32) greater than L-valine (17) greater than or equal to L-isoleucine (14) greater than L-allo-isoleucine (8); 14CO2 production was in the order L-valine (9) greater than L-isoleucine (6) greater than or equal to L-leucine (5) greater than L-allo-isoleucine (2). In variant (n = 5) as well as classical (n = 2) MSUD cell lines, branched-chain 2-oxo-[14C]acid release rates were generally comparable to the control values. As compared to the 14CO2 release in controls (= 100%), branched-chain 2-oxo acid dehydrogenase activity in MSUD fibroblasts was individually reduced and varied considerably between strains (residual activity 2-38%). Within individual strains, only small differences in the residual decarboxylation activity were observed in incubations with L-valine, L-leucine, and L-isoleucine. It was remarkably high, however, when L-allo-isoleucine was applied as a substrate. With the exception of L-allo-isoleucine, apparent total transamination rates of branched-chain L-amino acids were therefore distinctly lower in MSUD cells than in normal cells.

Metabolism of branched-chain amino acids in maple syrup urine disease.

Maple syrup urine disease (MSUD) is an autosomal recessive disorder. Impaired activity of the branched-chain 2-oxo acid dehydrogenase complex (BCOA-DH) causes accumulation of branched-chain L-amino (BCAA) and 2-oxo acids (BCOA) that can exert neurotoxic effects. MSUD presents as a heterogeneous clinical and molecular phenotype. Severity of the disease, ranging from classical to mild variant types, is commonly classified on the basis of indirect parameters, e.g. onset, leucine tolerance and/or residual enzyme activity in cells. Available information on BCAA turnover in vivo suggests that renal clearance is low and that the main route of BCAA disposal in MSUD is via protein synthesis, similar to healthy subjects. Information on BCAA oxidation is poor. In vivo oxidation rates have been assessed in a few studies in patients with claimed classical form of MSUD, using (stable) isotopically labelled L-leucine and both the (primed) continuous infusion and the oral bolus test approach. However, highly variable results have been obtained with both methods not only with respect to the number of patients exhibiting measurable leucine oxidation (range: 0%-100%; two to seven patients investigated) but also considering the extent of residual whole body leucine oxidation (range: < or = 2%-43% of control). Whether the different findings on whole body leucine oxidation actually reflect the variability of in vivo severity in classical MSUD as opposed to the measurements in cultured cells (generally < or = 2% of control), alternative pathways of leucine oxidation in some patients or were rather attributable to inadequate classification of patients or/and to inherent methodological problems remains to be clarified.

Coupled enzymatic assay for estimation of branched-chain L-amino acid aminotransferase activity with 2-Oxo acid substrates.

A convenient continuous spectrophotometric assay for estimation of branched-chain L-amino acid aminotransferase activity was established: Branched-chain 2-oxo acid-dependent transamination of L-glutamate was coupled-via 2-oxoglutarate-to L-aspartate aminotransferase plus L-malate dehydrogenase or to L-alanine aminotransferase plus L-lactate dehydrogenase as indicator systems. The rate of transamination can be monitored specifically by measuring the decrease in NADH absorbance at 334 nm over time. The method was applied, e.g., for evaluation of some kinetic properties of the rat heart (iso)enzyme.