CARBOHYDRATE METABOLISM

CARBOHYDRATE METABOLISM MGV - CLINICAL BIOCHEMISTRY

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

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

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

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)

HORMONAL CONTROL • A carbohydrate–rich 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

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

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

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

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,

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

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.

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.

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.

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 - Basedow’s disease - gluco-corticoid hormons - Cushing’s disease b) severe increase - pheochromocytoma (malignancy of adrenal medulla) with hypersecretion of epinephrine - glucagonom (tumours with pancreatic -cells) with hypersecretion of glucagon.

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

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.

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.

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.

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 lactation;galactosemia (associated with hypoglycemia); • Fructose: after fruit ingestion, pregnancy, lactation; fructose intolerance, essential fructosuria. • Pentose: chronic pentosuria (deficiency of the metabolism of glucogenetic amino acids).

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 it’s 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.

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).

IDENTIFICATION OF KETONE BODIES IN URINE BY LEGAL-IMBERT REACTION Principle: The most common method makes use of a reaction of sodium nitroprusside (Na2[Fe(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 children’s urine.

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).

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%

CARBOHYDRATE METABOLISM