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CARBOHYDRATE METABOLISM

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MGV - CLINICAL BIOCHEMISTRY CARBOHYDRATE METABOLISM BLOOD GLUCOSE HOMEOSTASIS Sources of glucose in the blood Diet Glycogenolysis (breakdown of glycogen ... – PowerPoint PPT presentation

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Title: CARBOHYDRATE METABOLISM


1
CARBOHYDRATE METABOLISM
  • MGV - CLINICAL BIOCHEMISTRY

2
BLOOD GLUCOSE HOMEOSTASIS
  • Sources of glucose in the blood
  • Diet
  • Glycogenolysis (breakdown of glycogen)
  • Gluconeogenesis (synthesis of glucose from
    noncarbohydrate substances)

3
1. DIET
  • Ingested carbohydrates
  • Digestible - starch or disaccharides which after
    digestion are transformed in glucose, galactose
    and fructose, that are absorbed, transported by
    the portal vein to the liver, where galactose and
    fructose are conversed in glucose
  • Nondigestible dietary fibers

4
2. THE LIVER
  • Its importance in glucose homeostasis consists
    in storage of the glucose as glycogen after
    food intake and maintaining the blood level by
    glycogenolysis and gluconeogenesis in the fasted
    state.
  • The hepatic uptake or output of glucose is
    controlled by the concentration of key
    intermediates and activity of enzymes
  • G enters the hepatocytes relatively freely
    compared with extrahepatic tissues
  • G phosphorilation is promoted by G-kinase with a
    lower affinity than hexokinase in extrahepatic
    tissues that is why little G is taken up by the
    liver at normal blood concentration compared to
    the more effective extraction by other tissues
    (brain) the activity of G-kinase is increased by
    hyperglycemia and the liver removes the G from
    the portal vein
  • Excess G is stored in the liver as glycogen

5
THE LIVER
  • In well-fed individuals hepatic glycogen stores
    represent 10 of the organ weight.
  • Glycogenolysis is the process by which the
    glucose is released from the liver the key
    enzyme is phosphorylase a, influenced by several
    hormones
  • Gluconeogenesis other compounds are converted
    in glucose
  • Lactate produced in the muscles and erythrocytes
    (anaerobic glycolysis), reconverted to glucose in
    the liver by the Cori cycle
  • Glycerol
  • Alanine formed in muscles by transamination of
    pyruvate (anaerobic glycolysis)

6
HORMONAL CONTROL
  • A carbohydraterich meal affects the release of
    hormones
  • Insulin release is
  • Stimulated by the gastrointestinal hormones
    (gastric inhibitory polypeptide (GIP), glucagon
    and aminoacids (arg, leu), vagal stimulation
  • Inhibited by somatostatin and sympathetic
    stimulation
  • Anabolic hormone
  • Stimulates G uptake by muscles and adipose tissue
  • Increases protein synthesis, glycogen synthesis
    and lipogenesis

7
HORMONAL CONTROL
  • Glucagon
  • Secretion stimulated by hypoglycemia,
    gluconeogenic aminoacids and inhibited by
    glucose, insulin, somatostatin
  • Stimulates glycogenolysis, gluconeogenesis,
    rising the glycemia

8
HORMONAL CONTROL
  • Growth hormone
  • Secretion stimulated by hypoglycemia
  • Action incresed glucose production in the liver,
    reduced uptake by some tissues
  • Adrenaline
  • Secretion stimulated by hypoglycemia
  • Action glycogenolysis, reduces insulin secretion
    resulting increasing the glucose concentration
  • Cortisol
  • Inhibits glycogenolysis, stimulates
    gluconeogenesis
  • They all stimulate lipolysis raising the NEFA
    production

9
INTERRELATION OF GLUCOSE, NEFA AND KETONE BODY
METABOLISM
  • During prolonged fasting and starvation the
    muscle, brain and other tissues oxidize
    alternative fuels as blood concentrations of
    these rise, reducing glucose utilization.
  • The supply of fatty acids is determined by the
    rate of release of NEFA from adipose tissue, this
    being controlled by the activity of
    hormone-sensitive lipase.
  • Insulin inhibits this enzyme (antilipolytic)
  • adrenaline, growth hormone, glucagon, cortisol
    are lipolytic

10
  • When carbohydrate supply is adequate small
    amounts of NEFA are released from the adipose
    tissue
  • When the carbohydrate supply is limited, greater
    amount of NEFA is released. They are transported
    bound with albumins in the blood, 30 ar
    extracted by the liver
  • Re-esterified to form TG
  • Metabolized by B-oxidation in mitochondria to
    form acetyl-CoA this can enter in Krebs cycle or
    form ketone bodies
  • Insulin inhibits and glucagon stimulates the
    mitochondrial carnitine-palmitoyl transferase I
    it enhances the transfer of FA into mitochondria,

11
DIABETES MELLITUS
  • Heterogeneous group of disorders characterized by
    hyperglycemia, glycosuria, abnormalities of lipid
    and protein metabolism
  • Clinical classification
  • Insulin-dependent diabetes mellitus (IDDM)
  • Non-insulin dependent diabetes mellitus (NIDDM)
  • Malnutrition-related DM
  • Diabetes associated with other disorders
  • Pancreatic diseases
  • Endocrine diseases
  • Congenital disorders
  • Gestational DM
  • Impared glucose tolerance

12
GLUCOSE IN THE BLOOD (GLYCEMIA)
  • Dosing the blood glucose depends on the reducing
    properties of this aldohexose. It is oxidized by
    hot alkaline copper solution, potassium
    ferricyanide solution. These methods give 10-20
    mg higher values because in the blood there are
    other reducing substances (gluthathion, ascorbic
    acid). Colorimetric methods are rapid and based
    on the reaction between the glucose and a
    chromogen (o-toluidine, anthrone).
  • Enzymatic methods are the most popular procedures
    because of their high specificity, rapidity of
    assay, use of small sample quantities (10 ?l) and
    easy of automation. The two enzymatic systems in
    most general use are those with hexokinase or
    glucose-oxidase as the first enzyme in a coupled
    reaction glucose dehydrogenase is used much less
    frequently.
  • No matter which method is used one must take
    precautions in sample collection to prevent
    glucose utilization by leukocytes, the glucose
    loss on standing in a warm room may be as high as
    10 mg/dl per hour. The decrease in serum glucose
    concentration is negligible if the blood sample
    is kept cool and the serum separated from the
    clot within 30 minutes of drawing. Otherwise,
    addition to the collection tube of 2 mg sodium
    fluoride per ml of blood to be collected prevents
    glycolysis for 24 hours without interfering with
    the glucose determination.

13
DOSING GLUCOSE IN THE BLOOD
  • COLORIMETRIC METHOD. CONDENSATION WITH
    o-TOLUIDINE
  • Principle Glucose condenses with o-toluidine
    when heated with acetic acid and forms a green
    chromogen whose absorbance (extinction) is
    measured at 630 nm. Ketohexoses and aldopentoses
    give a less intense colour their concentration
    is negligible (0.2-10 mg/L). Galactose in high
    concentration, as in galactosemia, interferes the
    glucose reaction in these cases an enzymatic
    method is prefered.
  • DETERMINATION OF SERUM GLUCOSE BY GLUCOSE OXIDASE
    METHOD
  • Principle This method employs glucose oxidase
    and a modified Trinder colour reaction, catalysed
    by peroxidase. Glucose is oxidized to D-gluconate
    by glucose oxidase with the formation of an
    equimolar amount of hydrogen peroxide. In the
    presence of peroxidase, 4-aminoantipyrine and
    p-hydroxybenzene sulfonate are oxidatively
    coupled by hydrogen peroxide to form a
    quinoneimine dye, intensely coloured in red. The
    intensity of colour in the reaction solution is
    proportional to the concentration of glucose in
    the sample.

14
DIAGNOSTIC IMPORTANCE OF GLYCEMIA
  • Reference values The results are not identical
    in whole blood in normal adult, a jeun, in all
    the methods used for analysis. That is why it is
    necessary to specify in the report the used
    method and the reference values for that specific
    method.
  •         o-toluidine method 65 -110 mg/dl
    3.6-6.1 mmol/L
  •         glucose oxidase 60 - 90 mg/dl
  • A single determination of glycemia has no
    diagnostical significance. The test has to be
    repeated.
  • Physiological variations
  • In new born the glycemia is decreasing in the
    first hours of life, but increases easily in a
    few days. In premature new born glycemia has low
    values 1.1-2.2 mmol/L.
  • In adult
  • low temperature, altitude, climate changing,
    emotional state, meals rich in carbohydrates,
    medication with atropine, pilocarpine determine a
    slight increase of glycemia.
  • muscular intense activity and fasting produce the
    decrease of glycemia.

15
  • Pathological significance
  • 1. Hyperglycemia (raised plasma glucose
    concentration)
  •        insufficient secretion of insulin
    (pancreatic ?-cells in islets)
  • a) primary diabetus mellitus
  • b) secondary to pancreatic or liver
    severe disease (acute pancreatitis, pancreatic
    neoplasm, pancreatectomy).
  •        hyperproduction of hyperglycemiant
    hormons
  • a) mild hyperglycemia
  • - growth hormone - acromegaly
  • - ACTH
  • - thyroidal hormones - Basedows disease
  • - gluco-corticoid hormons - Cushings
    disease
  • b) severe increase
  • - pheochromocytoma (malignancy of adrenal
    medulla) with hypersecretion of epinephrine
  • - glucagonom (tumours with pancreatic ?-cells)
    with hypersecretion of glucagon.

16
  • 2. Hypoglycemia (below 60 mg/100 ml 3.3 mmol/L)
  • a) Hormonal
  •    insulin excess
  • - overdosage of insulin in a diabetic or
    failure to eat after usual dosage
  • - excessive secretion in pancreas
    (pancreatic hyperplasia, insulinoma,
    sulfonylurea, leucine).
  • insufficiency of hyperglycemia hormones
  • b) Hepatic
  • - depletion of the liver glycogen stores
    (starvation, fasting, severe hepatocellular
    damage, phosphorus and CCl4 intoxication)
  • - - failure to release liver glycogen
    (genetic defects).
  • 3. Hereditary disorders (enzymatic defects) with
    reducing sugars in the urine
  •         - galactosemia (galactose-1-P uridyl
    transferase is lacking)
  •         - hereditary fructose intolerance
    (aldolase F-1.6-P to 2 triose-P)
  •         - fructose-1.6-diphosphatase deficiency
    (gluconeogenesis)
  • - essential fructosuria and pentosuria

17
GLUCOSE IN URINE (GLYCOSURIA)
  • Glucose is filtered through the glomerular
    membrane and totally reabsorbed in proximal
    tubule by an active transport.
  • Normally, the urine contains very small amount of
    glucose, less than 60 mg/L (100 mg/day).
  • When the glycemia is higher than 160-180 mg/dl,
    the ability of the tubular cells to transport the
    glucose is overwhelmed and the glucose is
    eliminated in urine (glycosuria or glucosuria).
  • In certain pathological conditions, other
    saccharides can exist in urine galactose,
    fructose, lactose, maltose, pentoses.

18
  • The identification of different urine saccharides
    is based on their reducing properties (except
    saccharose) of metal salts (Fehling, Benedict
    tests). The methods are less specific. Positive
    false results are given by increased
    concentrations of creatinine, uric acid, ascorbic
    acid, streptomycine, phenol compounds
  • When the presence of glucose in urine is noticed,
    the quantitative determination is necessary
  • Qualitative and semiquantitative methods use
    Clinitest tablets (Ames) or glucoseoxidase
    impregnated strips.
  • Quantitative tests use ortho-toluidine,
    hexokinase, glucose oxidase.

19
DIAGNOSTIC SIGNIFICANCE OF GLYCOSURIA
  • Reference values less than 60 mg/L (100 mg/day).
  • Physiological glycosuria appears after high
    glucose intake, physical effort.
  • Pathological significance
  • Glycosuria hyperglycemia
  • -  in diabetes mellitus (expressed in g/24
    hours)
  • - increased secretion of growth hormon, thyroidal
    hormones, glucocorticoids.
  • -  hepatic severe damage.
  • Glycosuria normal glycemia
  • -         renal diabetes (the tubular
    reabsorption is affected)
  • -         infectious diseases, nervous system
    affections
  • -         intoxication with morphine, atropine,
    lead.

20
GLUCOSE IN THE URINE
  • .
  • When other saccharides are present, they need to
    be identified.
  • Lactose exists physiologically in late
    pregnancy and lactation.
  • Galactose in infants during lactationgalactosemi
    a (associated with hypoglycemia)
  • Fructose after fruit ingestion, pregnancy,
    lactation fructose intolerance, essential
    fructosuria.
  • Pentose chronic pentosuria (deficiency of the
    metabolism of glucogenetic amino acids).

21
GLUCOSE IN THE URINE
  • .
  • When other saccharides are present, they need to
    be identified.
  • Lactose exists physiologically in late
    pregnancy and lactation.
  • Galactose in infants during lactationgalactosemi
    a (associated with hypoglycemia)
  • Fructose after fruit ingestion, pregnancy,
    lactation fructose intolerance, essential
    fructosuria.
  • Pentose chronic pentosuria (deficiency of the
    metabolism of glucogenetic amino acids).

22
KETONE BODIES IN URINE (KETONURIA)
  • Acetoacetic acid, ?-hydroxybutyric acid and
    acetone are classified as ketone bodies.
    Acetoacetic acid is the principal ketone body,
    synthesized by the liver mitochondria.
  • When there is insufficient oxalylacetic acid to
    derive the Krebs cycle for the formation of
    citrate and is used to synthesize the glucose,
    the acetate from acetyl-CoA is dimerized to yield
    aceto-acetyl-CoA.
  • ?-hydroxybutyrate dehydrogenase reduces much of
    acetoacetic acid to ?-hydroxybutyric acid.
  • Decarboxylase converts some of acetoacetate to
    acetone which is metabolized very slowly. Because
    its volatility, most evaporates through the lung
    alveoli.
  • Liver produces ketone bodies when the rate of
    acetyl-CoA formation exceeds of acetyl-CoA
    utilization by citric acid cycle.

23
KETONE BODIES IN URINE
  • Extrahepatic tissues (skeletal muscles, heart,
    renal cortex) utilize the ketone bodies (other
    than acetone) as a fuel. They oxidize
    ?-hydroxybutyrate to acetoacetate, then add
    CoA-SH by either of 2 routes to create
    acetoacetyl-CoA which is cleaved into 2
    acetyl-CoA able to enter Krebs cycle.
  • Food and Nutrition Board of U.S. recommends that
    the adult diet should contain al least 100 g or
    400 cal. carbohydrates daily to generate enough
    oxalylacetic acid to maintain TCA cycle and
    prevent ketosis. Carbohydrate defficiency causes
    protein waisting (much of dietary amino acids are
    converted via deamination and gluconeogenesis to
    glucose). The brain acquires a limited capacity
    for oxidizing ketone bodies after about 3 weeks
    of fasting, to protect against muscle waisting
    (gluconeogenesis from muscular proteins).
  •  

24
IDENTIFICATION OF KETONE BODIES IN URINE BY
LEGAL-IMBERT REACTION
  • Principle The most common method makes use of a
    reaction of sodium nitroprusside
    (Na2Fe(CN)5NO.2 H2O) and acetoacetate or
    acetone, under alkaline conditions a lavender
    colour is produced ?-hydroxybutyric acid does
    not react.
  •  Impregnated strips or sticks with reagent are
    introduced in urine for few seconds. By
    comparison with a colour chart, the concentration
    of acetoacetic acid and acetone is expressed as
  • -         negative
  • -         small 10 mg/dl
  • -         moderate 30 mg/dl
  • -         large 80 mg/dl
  • DIAGNOSTIC SIGNIFICANCE OF KETONE BODIES
  • Normally, the ketone bodies are not present in
    the urine of healthy individuals eating a mixed
    diet. (the reaction is negative)
  • Physiological values The ketone bodies may be
    present in childrens urine.

25
PATHOLOGICAL VARIATIONS
  • When there is high serum concentration of
    acetoacetate and ?-hydroxybutyric acid, the state
    is named ketonemia. It can overwhelme the blood
    buffers causing metabolic acidosis.
  • Ketonuria measures the acetone and acetoacetate
    detected by common hospital tests (may fail to
    detect ketonuria of ?-hydroxybutyric acid
    predominaters).
  •  
  • The ketosis (ketonemia associated with ketonuria)
    appears whenever
  • the rate of hepatic ketone body production
    exceeds the rate of principal utilization,
  • excessive amounts of fatty acids are catabolyzed
    and
  • the availability of glucose limited.
  • Hepatic overproduction is present in severe
    carbohydrate defficiency (diabetic ketoacidosis,
    alcoholic ketoacidosis, starvation ketosis) in
    this situation TCA cycle intermediates are
    depleted and this slows the entrance of
    acetyl-CoA into Krebs cycle. The acetyl-CoA
    carboxylase (the rate controlling enzyme of fatty
    acid synthesis) is inhibited by the absence of
    citrate, blocking another route of acetyl-CoA
    metabolism. Thus, acetyl-CoA accumulates in the
    liver and is excessively converted to ketone
    bodies.
  • The same conditions appear when the diet is poor
    in glucose but rich in lipids and proteins in
    gastrointestinal troubles (acute dyspepsia,
    toxicosis, vomiting during pregnancy, intense
    muscular effort).

26
GLYCOSYLATED HEMOGLOBIN
  • Used to monitor the diabetes therapy.
  • Three minor hemolobins are measured HbA1a,
    HbA1b, HbA1c, variants of HbA formed by
    glycosylation, an almost irreversible process in
    which glucose is incorporated in HbA. This
    reaction occurs with a constant rate during the
    120 days life span of an erythrocyte.
  • Thus, the glycosylated Hb reflects the average
    blood glucose level during the preceding 4-6
    weeks and offers information referring to
    long-term effectiveness of diabetes therapy.
  • Levels of glucose in the erythrocytes are more
    stable than plasma glucose.
  • Reference interval
  • HbA1a 1.6 of total Hb
  • HbA1b 0.8
  • HbA1c 5
  • Total glycosylated Hb 5.5-9 of total Hb
  • Pathologic results
  • Diabetes HbA1a and HbA1b 2.5-3.9 HbA1c 8-11.9,
    total 10.9-15.5
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