Percutaneous needle biopsy of skeletal muscle
Extensive investigation of the metabolism of normal and abnormal tissue requires a simple method for tissue sampling. The percutaneous muscle biopsy technique developed and introduced by Bergstrom in the 1960s provides a simple and repeatable sampling method Bergstrom 1962, 1975 , which has made skeletal muscle one of the few tissues suitable for direct studies of structure and metabolic functions in human subjects. The percutaneous muscle biopsy procedure has been used widely to study many...
Info Vbo
1,608 1,610 1,612 1,614 1,616 1,618 m z Figure 10.4 2H2O-Proteomics Panel A demonstrates that a steady-state labeling of body water can be achieved using 'primed-infusion' of 2H2O on day 0 mice were given a bolus of 2H-labeled saline 20 ul per g body weight and then maintained on 5 2H-labeled drinking water days 0 to 10 . Mice had ad libitum access to food and were killed on various days n 3 per day and blood was collected. The rate of albumin synthesis was determined by fitting the labeling...
Spontaneous baroreflex sensitivity
The development of a technique based on servoplethysmomanometry that measures blood pressure in the finger on a beat-to-beat basis Pei z et al. 1976 has expanded the diagnostic spectrum in diabetic patients with CAN. This method Finapres is increasingly becoming an integral constituent in the assessment of ANS function, allowing the assessment of neural modulation of the sinus node by arterial baroreceptors. In the past decades, the baroreceptor-cardiac reflex sensitivity BRS has been...
Biopsy procedure and sample handling
All research involving skeletal muscle biopsies must be approved by an institutional review board ethics committee. The subject must be fully informed about the study and the potential risk and discomfort - this includes information about the potential establishment of muscle cell cultures and the long-term storage and use of the obtained biological material. For subjects undergoing skeletal muscle biopsies, we recommend careful history-taking and physical examination to exclude current...
Glutamination
Phenylacetate is conjugated to glutamine in primates and the conjugate is excreted in urine Thierfelder amp Sherwin 1915 James et al. 1972 . The enzyme activating the phenylac-etate to its coenzyme A derivative and catalyzing the conjugation, acyl CoA L-glutamine N-acyltransferase, is present in the liver and kidney Moldave amp Meister 1957 . Glutamine via glutamate is formed from a-ketoglutarate, an intermediate in the Krebs cycle. Phenylacetate is normally found in small quantity in human...
Effects of glycogen cycling
The term 'glycogen cycling' refers to a futile cycle between glycogen and glucose-1-phosphate G1P . Since G1P is also in rapid exchange with G6P, glycogen cycling influences the 2H-enrichment of G6P by trapping labeled G6P molecules in glycogen and replacing them with unlabeled equivalents. Under fasting conditions, glycogen cycling will therefore diminish G6P enrichment from gluconeogenic precursors, hence the gluconeogenesis contribution will appear to be reduced while the glycogenolysis...
Arteriovenous balance measurements using isotope tracers
The regulation of protein homeostasis can be studied in specific anatomical locations by measuring the plasma or blood flow and the concentration difference of amino acids across limbs and or tissue beds of interest. A simple equation that describes net protein balance is flow x concentrationaltery concentrationvein 10.8 where concentration is the concentration of an amino acid in a blood vessel ideally one should calculate the balance of all amino acids. This approach is based on principles of...
Homa
As with the HOMA method for insulin resistance see Chapter 3 , the calculation of the HOMA P-cell function index HOMA-B requires only fasting glucose and insulin measurements. A model-based version of the method HOMA2, http www.dtu.ox.ac.uk homa web search keywords homa calculator,site dtu.ox.ac.uk is recommended, although a formula is also available HOMA-B constant X fasting insulin fasting glucose - 3.5 Matthews et al. 1985 . The advantages of the HOMA2 computer index have not been...
The cell culture principle of human myotubes
Satellite cells can be isolated from muscle biopsy samples by enzymatic digestion Yasin et al. 1977 Blau amp Webster 1981 or by migration from muscle tissue explants Askanas amp Engel 1975 . We and most other laboratories use the former principle. The initial myogenic purity after enzymatic digestion is high gt 95 but it decreases easily through fibroblast overgrowth and as satellite cells differentiate into myotubes. The purity and the differentiation degree of obtained myotubes cultures can...
Glucuronidation
Glucuronidation is the most widespread process for the conjugation of drugs in the body Dutton 1980 . It functions mainly in the liver. The conjugation to glucuronic acid increases the drug's water solubility, resulting in more rapid urinary excretion. Acetaminophen is excreted both as its glucuronide and sulfate conjugates, the former in larger percentage the higher the dose of the acetaminophen. The enzyme catalyzing its coupling is UDPglucuronyl transferase, acetaminophen UDPglucuronic acid...
Dual Energy Xray Absorptiometry DXA
Measurement of body composition by dual energy X-ray absorptiometry is based on differences in the absorption of X-rays by various atoms. The different molecular compositions of fat, lean tissue and bone material result in different X-ray absorption in these tissues. X-rays of 40 keV and 70 keV, produced by tungsten X-ray tubes and filtered by rare-earth material filters, are used for the measurement of localised tissue X-ray attenuation in planar projection. The fat and lean mass composition...
Calculation of whole body glucose metabolism M value
Provided that endogenous glucose output is zero or at least negligible, the glucose infused equals the glucose being translocated out of the glucose space i.e. glucose metabolised, M value during steady state conditions of hyperinsulinaemic euglycaemia DeFronzo Glucose flux into the glucose space 2 mg-kg-1-min-1 Glucose flux out of the glucose space 2 mg-kg-1-min-1 Figure 4.3 Schematic diagram of postabsorptive glucose metabolism in healthy humans. Under postabsorptive conditions the glucose...
References Fkk
Armstrong DT, Steele R, Altszuler N, Dunn A, Bishop JS, DeBodo RC 1961 Regulation of plasma free fatty acid turnover Am J Physiol 201, 9-15 Barrows BR, Timlin MT, Parks EJ 2005 Spillover of dietary fatty acids and use of serum nonesterified fatty acids for the synthesis of VLDL-triacylglycerol under two different feeding regimens Diabetes 54, 2668-2673 Barter PJ, Nestel PJ 1972 Plasma free fatty acid transport during prolonged glucose consumption and its relationship to plasma triglyceride...
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In the original work of DeFronzo etal. 1979 the computation of rates of whole body glucose metabolism M during the clamp was calculated from the means of 20-min periods by the following equation where GIR is the glucose infusion rate, SC the space correction and UC the correction for urinary glucose loss. If M is calculated for 20-min periods as recommended, the plasma glucose concentrations at the beginning and the end are computed, for the calculation of the space correction, according to the...
Coupling nitrogen balance and isotope tracers
Measurements of total nitrogen intake and output can be used to calculate protein nutritional status Leverton amp Gram 1949 Hegsted 1978 . This approach is based on principles of chemical mass-balance, and although practical matters concerning collecting total nitrogen waste make this method difficult to implement, measurements of nitrogen balance generally permit a reliable determination of protein homeostasis. Use of the method requires special consideration since urinary nitrogen may occur...
Advantages and limitations of needle biopsy
The needle biopsy circumvents many of the disadvantages of the open muscle biopsy, which include higher costs, the need for general anaesthetic, increased scarring and the inconvenience of repeated biopsies Goldberger et al. 1978 Edwards et al. 1980, 1983 . The fact that many of our patients and control subjects have participated in several studies involving muscle biopsies emphasises that this technique is easy to learn, repeatable and relatively atraumatic. It is a safe procedure that is...
References Ewt
Burgess SC, Weis B, Jones JG, Smith E, Merritt ME, Margolis D et al. 2003 Analy Biochem 312, 228-34 Chandramouli V, Ekberg K, Schumann WC, Kalhan SC, Wahren J, Landau BR 1997 Am J Physiol 273, E1,209-15 Chandramouli V, Ekberg K, Schumann WC, Wahren J, Landau BR 1999 Am J Physiol 277, E717-23 Jones JG, Barosa C, Gomes F, Mendes AC, Delgado TC, Diogo L et al. 2006a J Carbohydr Chem 25, 203-17 Jones JG, Carvalho RA, Sherry AD, Malloy CR 2000 Anal Biochem 277, 121-6 Jones JG, Fagulha A, Barosa C,...
Hypoglycaemia questionnaires
In hypoglycaemia research, it may be important to document the quality as well as the quantity of hypoglycaemia. Problematic hypoglycaemia may be described in terms of the frequency of severe hypoglycaemia and there are at least three validated questionnaire-based assessments of subjective awareness of hypoglycaemia. Clarke's questionnaire is a set of questions aiming at self-assessment of hypoglycaemia awareness. A scoring system stratifies the individual as having impaired awareness or being...
Magnetic Resonance Imaging MRI and spectroscopy
Positioning of measured samples in strong homogeneous magnetic fields forces protons within the sample to align according to the direction of the magnetic field and thus create a macroscopic magnetisation of the sample. Interaction of spins with externally applied radiofrequency RF waves at the resonant frequency changes the magnitude and direction Figure 13.3 Assessment of Abdominal Fat Storage by Computed Tomography CT Representative cross-sectional abdominal CT scans of a lean A and an obese...
Nomenclature of glycaemic targets of clamp tests
In healthy subjects an insulin clamp performed at fasting plasma glucose concentration is termed an euglycaemic insulin clamp test. In general, when the target of plasma glucose concentration is above the fasting level, this is termed a hyperglycaemic insulin clamp test. The latter should not be mixed up with the hyperglycaemic glucose clamp test, which is performed without concomitant infusion of insulin and which can be used to assess endogenous insulin secretion in response to glucose...
Hypoglycaemia Recording Form
Todays date dd mm yy Time of hypo Did you have any warning symptoms Q YES Q NO If yes, please indicate which, if any, of the following occurred tick more than one box if necessary Confusion Sweating Drowsiness Weakness Dizziness Warmness Difficulty speaking Pounding heart Inability to concentrate Shivering Unsteady on feet Yellow vision Double vision Blurred vision Hunger Thirst Nausea Anxiety Tiredness Tingling lips Trembling Headache Abdominal pain Other specify How did the intensity of these...
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Stephanie A. Amiel BSc, MD, FRCP, RD Lawrence Professor of Diabetic Medicine, Gene and Cell Based Therapy, Diabetes Research Group, Diabetes, Endocrinology and Metabolism, King's College London School of Medicine, London, UK Henning Beck-Nielsen MD, DMSc, Head of the Department of Endocrinology, Odense University Hospital, Professor of Endocrinology, University of Southern Denmark, and Head of the Danish PhD School of Molecular Metabolism, Odense, Denmark Ayad Al-Bermani MD, Specialist...
Blood sampling site arterial venous or arterialised venous blood sampling
Matched hyperinsulinaemia rates of whole body glucose metabolism can only be directly compared among individuals if the concentration of glucose perfusing their glucose utilising tissues is the same Bergman etal. 1985 . The concentration of plasma glucose physiologically declines during the perfusion of glucose utilising tissues. In addition, pancreatic insulin secretion is regulated by the arterial blood glucose concentration. Early studies applying the glucose clamp technique measured the...
Vldl
Very low density lipoproteins VLDL are of interest for two reasons. First, they are the obligate precursor of low density lipoproteins LDL Dietschy et al. 1993 . Second, together with chylomicrons, VLDLs represent the major vehicle for transport of triglycerides in the circulation. VLDL triglyceride secretion by the liver is controlled to some extent by the supply of FFA to that tissue Lewis et al. 1995 , with additional acute regulation by insulin Sparks amp Sparks 1994 . Hepatic VLDL...
Pancreatic clamp tests
In order to achieve a maximal possible suppression of endogenous insulin, glucagon and growth hormone release during hyperinsulinaemic clamp tests, a concomitant infusion of somatostatin can be applied Gottesman etal. 1982 Best etal. 1983 Basu etal. 2000 . Glucose clamp tests applying an infusion of somatostatin are commonly referred to as pancreatic clamp tests. Most investigators start the somatostatin infusion a few minutes before or simultaneously with the start of the insulin infusion. The...