Diseases in Saccharide Metabolism

Biochemistry of Carbohydrates

Functions

Saccharide

• energy source
• precursors of essential metabolites for use in synthetic processes
• components of nucleic acids and of some coenzymes
• essential lipid metabolism

Digestion

• Saliva (amylase, maltase)
• stomach (HCl, pH 2-2.5) destroys the enzymatic action
• small intestine enzymes of the pancreatic juice and of the succus entericus
  • starch and glycogen into glucose by amylase and maltose
  • lactose into glucose and galactose by lactose
  • saccharose into glucose and fructose by sucrase
  • glucose, galactose and fructose are principle forms of carbohydrates in which absorption occurs in monogastric animals

Absorption

• the mucosa of the small intestine into portal circulation
  • facilitated diffusion
  • sodium-dependent active transport
• glucose and galactose absorbed rapidly by both methods
• fructose absorbed about half the rate of glucose with a portion being converted to glucose in the process
• the active absorption of glucose across the intestinal mucosa is connected with phosphorylation in the mucosal cell. The phosphorylated sugar is then transferred across the mucosal cell, re-hydrolysed, and free glucose appears in the portal circulation for transport to the liver

Metabolism of Absorbed Carbohydrates

Hexose Level in Blood

Liver Cells

• permeable to the absorbed glucose
• facilitated by glucose transporter proteins within the plasma membrane (GLUT-2)
• the fate of the absorbed glucose depends on the organism’s demands
  • metabolised or stored in the liver
  • released into blood circulation for the needs of other organs

Fate of Hexoses

• during and after feeding
  • absorbed hexoses are converted to glucose by the liver and stored in glycogen or as fat
• during fasting
  • glucose is supplied from the liver into circulation by two metabolic pathways
  • gluconeogenesis
  • glycogenolysis

Glucokinase vs Hexokinase

• glucokinase
  • high Km (=2x10^-2 mol/l)
  • low affinity for glucose
  • inducible enzyme
• hexokinase
  • low Km (=5x10^-5 mol/l)
  • high affinity for glucose
  • is not an inducible enzyme

Regulation of Glycogenolysis

• epinephrine (adrenaline) - increases glucose due to stress reaction
  • acts on both liver and muscle glycogen
• glucagon - maintain physiological levels of glucose
  • acts only on liver glycogen
• other polypeptide hormones
  • via adenylate cyclase and cAMP
  • ACTH (adrenocorticotropic hormone)
  • LH (luteinising hormone)
  • TSH (thyroid stimulating hormone)
  • MSH (melanocyte stimulating hormone)
  • T3 (tri-iodothyronine)
  • via tyrosine kinase activity
  • insulin

Glucose Transport Proteins

• GLUT-1 (brain, RBC, placenta, kidney)
• GLUT-2 (liver, pancreas)
• GLUT-3 (brain)
• GLUT-4 (skeletal, cardiac muscle, adipose tissue)
• GLUT-5 (small intestine; also transports fructose)

Glycaemia = blood glucose concentration lower in polygastric animals when compared to monogastric animals

Role of the Liver

• the membrane transfer system is the rate-limiting in peripheral tissues which are sensitive to insulin (muscle, adipose)
• this mechanism is not rate limiting in the liver because glucose diffuses freely across the liver membrane
• at blood glucose level of 8.33 mmol/l the liver does not take up or supply glucose to the circulation
• this level is termed steady state or the glucostatic level (glucostat)
• since the fasting blood glucose is about 5 mmol/l the liver supplies glucose throughout the day except for postprandial period when blood glucose is greater than 8.33 mmol/l

Glucose Tolerance

When administered orally to a normal animal

• I. Phase: absorptive - the rate of glucose entry into circulation exceeds the rate of removal and the blood glucose rises. As the blood glucose rises, hepatic glucose output is inhibited and the release of insulin from the pancreas is stimulated. In 30-60 minutes, the peak level of blood glucose is reached. After it, blood glucose begins to fall
• II. Phase: removal of the glucose excess - the rates of removal now exceed those of entry, hepatic glucose output decreases and the blood glucose falls rapidly until it reaches baseline level
• III. Phase: hypoglycaemic phase - when blood glucose reaches baseline level, it continues to fall for a short time. Then it returns to its baseline level

Storage of Glycogen

• glycogenesis
• α-1,4 and α-1,6 glycosidic bonds

Determination of Saccharides in Animals

Biochemical Methods

take into account glucose breakdown (glycolysis) by RBC which takes place very rapidly, approximately 10% per hour loss, at room temperature. The plasma or serum must be separated from RBC within 1/2 hour

• Direct Methods - measurement of actual glucose concentration using spectrophotometry; adapted for use by automatic analysers
  • toluidine method
  • is not a specific method because it detects galactose and mannose in addition to glucose
  • the amount of these saccharides in blood is not significant and the values are taken as “true glucose”
  • hexokinase measurement
  • the amount of NADPH is measured spectrophotometrically and serves for calculation of glucose concentration
  • glucose + ATP (hexokinase) → glucose-6-P + ADP
  • glucose-6-P + NADP (glucose-6-P-dehydrogenase) → 6-GP + NADPH
  • the amount of reduced NADPH is measured spectrophotometrically and serves for calculation of glucose concentration
  • glucose dehydrogenase method
  • glucose + NAD (glucose dehydrogenase) → gluconolactone + NADH
  • NADH is determined spectrophotometrically
  • glucose oxidase method
  • glucose (glucose oxidase) → gluconic acid + H2O2
  • H2O2 + ABTS (peroxidase) → H2O + green colour
  • the intensity of green colour is proportional to the glucose concentration and is determined spectrophotometrically
  • ABTS (abbr. di-amonium 2,2´-azino-bis (3-ethylbenzotiazoline-6-sulphonate)
• Indirect Methods - monitoring blood glucose methods using so-called glycated proteins
  • hemoglobin A1c test
  • in dog, HbA1c has a half-life of 60 days so it reflects the average blood glucose 2 months prior to sampling
  • in cat, HbA1c has a half-life of 40 days, therefore it is possible to determine blood glucose concentration back to 6 weeks prior to sampling
  • determination of blood glucose concentration using glycated proteins needs sophisticated laboratory equipment (HPLC, immunoassay, electrophoresis)
  • fructosamine test
  • albumin: half-life 7-9 days
  • fructosamine - an indicator of average blood glucose from the previous 2 weeks
  • the advantage of detecting changes in glucose control more quickly than HbA1c
  • a colourimetric assay readily performed in clinical laboratory
    • dog: 1.7-3.38 mmol/l
    • cat: 2.19-3.47 mmol/l
  • Principle
  • glucose molecules irreversibly bound to proteins are known as glycated proteins
  • when the protein of the complex is haemoglobin, the product in haemoglobin A1c
  • when the protein of the complex is albumin, the product is fructosamine
  • the binding of glucose to the protein is firm and constant over the lifespan of a particular protein
  • glycated proteins reflect the average blood glucose over the half-life of the protein
  • they offer a means for evaluation of long-term blood glucose in diabetics

Clinical Methods

• tolerance tests

  • glucose tolerance test
  • oral glucose tolerance test (OGTT)
    • dog: a test meal consisting of 4 g glucose/kg body weight mixed with a few grams of meat
    • a fasting blood sample is taken
    • the test meal is given
    • blood samples are taken at 30-min intervals for 3 hours
    • interpretation:
      • 1 hr after feeding the maximal glucose level is reached (6.6-7.7 mmol/l)
      • after 2-3 hr the glucose level returns to the fasting level (3/6-5/3 mmol/l)
    • NB: may be simplified by taking a single sample at 2 hr after giving glucose
      • normal blood glucose level 2 hr postprandially indicated diabetes is unlikely
      • hyperglycaemia at 2 hr postprandially indicated diabetic curve and a COMPLETE TEST SHOULD BE DONE

• Intravenous Glucose Tolerance Test (IVGTT)

  • At this method
  • animal is not overloaded with glucose
  • infusion can be given within the time limits
  • blood glucose level is high enough to give a maximal insulin response
  • urinary loss of glucose is minimal
  1. after a standard fasting (12 – 16 h) (overnight) a zerotime heparinized blood sample is taken
  2. 0.5 g glucose/kg body weight is infused in 30 s intervals
  3. blood samples are subsequently taken at 5, 15, 25, 35, 45
     and 60 min
  4. plasma glucose is determined in the all samples, results
     are plotted semilogarithmically
  5. reading: time required for the glucose concentration fall by
     one-half (T1/2) is estimated between 15 – 45 minutes
     postinfusion from graph
  6. The normal T1/2 in dog is 25 min
     the diabetic dog has a longer T1/2
  • amount of glucose that could be ingested by an animal without producing glucosuria
  • an animal with an increased glucose tolerance has a limited rise and rapid fall in blood glucose (it can tolerate extra glucose)
  • an animal with decreased glucose tolerance has an excessive rise in glucose and a slow return to its baseline, i.e. cannot tolerate extra glucose - typical diabetic type of GT curve
  • in dog, for optimal results, the animal must be placed on a standard diet of 100-200 mg carbohydrate plus fat and protein per day 3 days prior to performance of the test
  • insulin tolerance test
  • test measures
    • sensitivity of the blood glucose level to a test dose of insulin
    • the response of the animal to insulin-induced hypoglycaemia
  • interpretation: normally, blood glucose level falls to 50% of its fasting level in 20-30 minutes, and returns to its fasting level in 1.5-2 hr
  • two types of abnormal responses are seen
    • insulin resistant (insensitive) response - blood glucose level does not fall by 50% or requires more than 30 min to reach the maximum hypoglycaemic level (in hyperpituitarism and adrenals)
    • unresponsive hypoglycaemia - the hypoglycaemia is prolonged and fails to return to the fasting level in 2 hr (observed in hyperinsulinism, hypopituitarism, hypoadrenalism)

  Disorders of Carbohydrate Metabolism

  Diabetes Mellitus

  • in many species
  • most frequently found in dogs and cats
  • for both species, obesity predisposes development of diabetes mellitus
  • some other factors: pancreatitis, infections, stress, oestrus
  • the fundamental defect in diabetes mellitus: absolute or relative lack of insulin resulting in an inability to utilise glucose
  • based on the insulin response during IVGTT it is possible to distinguish
  • Type I (loss of pancreatic β-cells) - some viral infections, autoimmune diseases
    • in the affected dogs, very low initial insulin level is found and no insulin response is observed to the administration of glucose
  • Type II (due to receptor defect or deficiency)
    • normal to high initial level of insulin is observes, however, without response to the administration of glucose
  • Type III
    • characterised by normal initial insulin level, normal to delayed insulin response to glucose and prolonged return of insulin return to the normal value after 60 min
  • due to insulin deficiency, the glucose intolerance is observed in all three types of diabetes

  Hyperinsulinism (Hypoglycaemia)

  • It is known to be due to persistent hyperactivity of the pancreas as the result of insulin-secreting islet cell tumours
  • occurs in: dogs, children
  • characterised by a persistent hypoglycaemia with periods of weakness, apathy, convulsions, and coma
  • diagnosis
  • hypoglycaemia of < 3 mmol/l
  • hyperinsulinaemia of > 20 μU/ml
  • after administration of glucose, the symptoms are relieved

  Development in piglets

  • at birth, the blood glucose level is > 6 mmol/l
  • unless the pig is fed, its blood glucose drops rapidly to hypoglycaemic level within 24-36 hours. If the piglet suckles, its ability to withstand starvation progressively increases. A 10-day-old piglet can starve up to 3 weeks before symptoms of hypoglycaemia occur (apathy, weakness, convulsions, coma, and death)
  • reasons of starvation
  • in mother: agalactia, metritis
  • in piglet: anaemia, infections
  • probable basis of newborn baby pigs hypoglycaemia
  • gluconeogenic mechanisms (glucose synthesis) are underdeveloped and gluconeogenic enzymes must be induced by feeding within after birth to reach their maximal activities