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