Clinical Chemistry

1. Clinical Chemistry Carbohydrate metabolism Mohammed Al-Zubaidi, PhD

2. CLINICAL BIOCHEMISTRY AND METABOLIC MEDICINE • By Martin A Crook • Eighth eidition

3. Functions of extracellular glucose • The important function of glucose is as a source of body (tissue) energy • All tissues can utilize glucose, among them brain, which is highly dependent on extracellular glucose as an energy supplier ( glucose impair cerebral function, lead to irreversible neuronal damage)

4. Why brain highly dependent on glucose? • Can not synthesis glucose • Can not store glucose in significant amount • Can not metabolize substance other than glucose and ketones (under physiological conditions, ketones are of little importance as an energy source) • Can not extract glucose from the ECF at low concentrations because entry into brain cells is not facilitated by insulin

5. Plasma glucose homeostasis Maintenance of plasma glucose concentrations within the range of 4-10 mmol/L depends on the balance between the glucose entering cells from ECF and that leaving them into ECF • Hormonal control: • Insulin • Glucagon • Somatostatin • Other hormones • The liver

6. Hormonal control • Insulin: • when plasma glucose 5 mmole/L acting via the glucose transporter 2 stimulates insulin release from the pancreas ᵦ-cell. • ᵦ-cell produce proinsulin (consist of 51 amino-acid poly peptide insulin and a linking peptide)

7. Hormonal control • Splitting of the peptide bonds by prohormone convertases releases via intermediates (mostly 32-33 split proinsulin) equimolar amounts of insulin and c-peptide into ECF. • Transport of glucose into liver cells is insulin independent (reducing the intracellular glucose concentration via indirectly promote the passive diffusion of glucose into liver cells)

8. Hormonal control 2. Glucagon • Glucagon is a single chain polypeptide synthesized by alpha-cells of pancreatic islets. • Glucagon secretion stimulate by hypoglycemia. • Glucagon enhance glycogenolysis and gluconeogenesis.

9. Insulin AND Glucagon hormones

10. Hormonal control 3- Somatostatin • It is peptide hormone release from the D cells of the pancreas. • Somatostatin inhibits insulin and growth hormone release. 4- Other hormones: • Hyperglycemic action (including, growth hormone (GH), glucocorticoid, adrenaline (epinephrine) and glucagon) even if there is no increase in secretion rates. • Secretion of these hormone so-called counter-regulatory hormones may increase during stress and in patients with acromegaly (GH), cushing’ssyndrom (glucocorticoid) or phaeochromocytoma (adrenalin).

11. 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: • Glucose enters the hepatocytes relatively freely compared with extrahepatic tissues. • Glucose phosphorylation is promoted by Glucose kinase with a lower affinity than hexokinase in extrahepatic tissues; that is why little Glucose 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 Glucose from the portal vein. • Excess glucose is stored in the liver as glycogen

12. The liver • The liver cells can store some of the excess of glucose as glycogen and the rate of glycogen synthesis (glycogenesis) may be increased by insulin secretion. • The liver can convert some of excess glucose to fatty acids which are ultimately transported as triglyceride in VLDL and stored in adipose tissue. • Gluconeogenesis – other compounds are converted into glucose: • Lactate • Glycerol • Carbon chains resulting from deamination of certain amino acid (mainly alanine)

13. The liver • The liver contain glucose-6-phosphatase, which hydrolyze G6P derived from glycogenolysis or gluconeogenesis, releases glucose and help maintain extracellular glucose level. • Hepatic glycogenolysis stimulated by glucagon hormone in response to fall in the plasma glucose; and by catecholamines (adrenaline or nor adrenaline). • During fasting, adipose tissue will release fatty acids as a consequence of low insulin, the liver converts these fatty acids to ketones. • Carbon chains of some amino acids may also converted to ketones • Ketones can be used as energy source by other tissues, including brain, when plasma glucose level is low.

14. Other organs • The other tissue capable of gluconeogensis is renal cortex, by converting G6P to glucose. • The gluconeogenesis capacity of the kidney is important in hydrogen ion homeostasis and during prolonged fasting. • Other tissues, such as muscle can store glycogen but cannot release glucose from cells because they don’t contain glucose-6-phasphatase, so can only use it locally. Glycogen in theses tissues will not play any important role in maintaining the plasma glucose level.

15. Ketosis • Adipose tissue triglyceride is the most important long-term energy store in the body. • Increased use of fat stores during prolonged fasting is associated with ketosis. • Adipose tissue cells acting in conjunction with the liver, convert excess glucose to triglyceride and store it in this form rather than glycogen. • The components of triglyceride (fatty acid & glycerol) are both derived from glucose, fatty acids from the glucose entering hepatic cells and glycerol from that entering adipose tissue cells.

16. Ketosis • During prolonged fasting and starvation (exogenous glucose is unavailable and the plasma insulin concentration is therefore low), endogenous triglycerides are converted to free non-esterified fatty acids (NEFAs) and glycerol by lipolysis. • Both NEFAs and glycerol transported to the liver in plasma, NEFA being protein bound (to albumin), while glycerol enter the hepatic gluconeogenic pathway at the trios phosphate stage; the glucose synthesized can be released from these cells and lead to minimize the fall in glucose concentrations

17. Ketosis

18. Ketosis • Most tissues, other than brain, can oxidize fatty acids to acetyl CoA, which can then be used in TCA cycle as an energy source. • When the rate of synthesis exceeds its used, hepatic cells produce acetoacetic acid by condensation of two molecules of acetyl CoA; acetoacetic acid can be reduced to beta-hydroxybutyric acid and decarboxylated to acetone. • These ketones can be used as an energy source by brain and other tissues when glucose is relatively short supply.

19. Ketosis • During fasting, fat stores are the main energy source and ketosis occurs. • Mild ketosis may occur after as little as 12 h of fasting • Metabolic acidosis is not usually detectable after short fasting, but after long periods, more hydrogen ions may be produced.

20. Pathological lactic acidosis • Lactic acid produced by anaerobic glycolysis, may either be oxidized to CO2 and water in the TCA cycle or be reconverted to glucose by gluconeogenesis in the liver. • Pathological accumulation of lactic acid may occur because: - production is increased by an increased rate of anaerobic glycolysis - Use is decreased by impairment of the TCA cycle or impairment of gluconeogenesis

21. Pathologic lactic acidosis • Hypoxia increases plasma lactate concentration because: - The TCA cycle cannot function anaerobically and oxidation of pyruvate and lactate to CO2 and water is impaired. - Hepatic and renal gluconeogenesis from lactate cannot occur anaerobically. - Anaerobic glycolysis is stimulated because the falling ATP levels cannot be regenerated by the TCA cycle under anaerobic conditions.

22. Pathological lactic acidosis • The combination of impaired gluconeogenesis and increased anaerobic glycolysis converts liver from an organ that consumes lactate and H+ to one that generates large amounts of lactic acid. Sever hypoxia for example following cardiac arrest cause marked lactic acidosis.

23. Hyperglycemia and diabetes mellitus • Hyperglycemia may be due to: • Intravenous infusion of glucose-containing fluids • Sever stress (transient effect) such as trauma, myocardial infarction or cerbrovascular accidents. • Diabetes mellitus or impaired glucose regulation.

24. Diabetes mellitus • DM is caused by an absolute or relative insulin deficiency. • It has been defined by WHO, on the basis of laboratory findings, as a fasting venous plasma glucose concentration of 7.0 mmol/l or more (more than one occasion in the presence of diabetes symptoms) or a random venous plasma glucose concentration of 11.1 mmol/l or more. Sometime an oral glucose tolerance test (OGTT) may be required to establish the diagnosis.

25. Classification of DM I. Type 1 diabetes mellitus (insulin-dependent diabetes mellitus) • Due to destruction of beta cells of pancreatic islets • Consequence: absolute deficit of insulin • Present during childhood and adolescence • Insulin therapy is essential A- subtype: induced by autoimmunity processes. B- subtype: idiopathic diabetes mellitus. There is also LADA (Latent autoimmune diabetes of adults), sometime called slow-onset type 1 diabetes

26. Classification of DM II. Type 2 diabetes mellitus (non insulin-dependent diabetes mellitus) • The disorder ranging from mainly insulin resistance with relative insulin deficiency to a predominantly secretory defect with insulin resistance. • Insulin may sometimes be needed • Onset is most usual during adult life • There is familial tendency and an associated with obesity.

27. III. Other specific types of diabetes mellitus A variety of inherited disorders may responsible for the syndrome, either by reducing insulin secretion or by causing relative insulin deficiency because of resistance to its action or of insulin receptor defects, despite high plasma insulin concentration. • Genetic defect of beta-cell function • Maturity-onset diabetes of the young (MODY): • MODY 1: mutation of hepatocyte nuclear factor (HNF4A) gene • MODY 2: mutation of glucokinase gene • MODY 3: mutation of HNF1A gene Some cases are thought to be point mutation in mitochondrial DNA associated with DM and deafness

28. III. Other specific types of diabetes mellitus • Genetic defect of insulin action: Type A insulin resistance (insulin receptor defect), for example leprechaunism, lipoatrophy and Rabson-Mendenhall syndrom • Insulin deficiency due to pancreatic disease: 1- Chronic pancreatitis 2- Pancreatectomy 3- Haemochromatosis 4- Cystic fibrosis • Endocrinopathies: Relative insulin deficiency, due to excessive GH (acromegaly), phaeochromocytoma, glucocorticoid secretion (Cushing’s syndrom)

29. III. Other specific types of diabetes mellitus • Drugs: 1- thiazide diuretics 2- Interferon alpha 3- Glucocorticoids • Infections: 1- Septicaemia 2- Congenital rubella 3- Cytomegalovirus

30. III. Other specific types of diabetes mellitus • Rare form of autoimmune-mediated diabetes 1- Anti-insulin receptor antibodies 2- Stiff man syndrome, with high level of GAD autoantibodies • Genetic syndrome associated with diabetes 1- Down’s syndrome 2- Turner’s syndrome 3- Klinefelter’ssyndrom 4- Myotonic dystrophy

31. IV. Gestational diabetes mellitus • Women at high risk for GDM including: previously given birth to a high-birth weight baby, obese, family history of DM, high-risk ethnic groups (black or South Asian). • Women should screened at the earliest opportunity and, if normal, retested at about 24-28 weeks, as glucose tolerance progressively deteriorates throughout pregnancy. • Women have GDM If fasting venous plasma glucose ≥ 7.0 mmol/l and/or random measurement ≥ 11.1 mmol/l. • Six weeks post partum, the woman should reclassified with repeat OGTT

32. Impaired glucose tolerance (IGT) • Fasting venous plasma glucose concentration < 7.0 mmol/l • 2 h after OGTT, plasma glucose between 7.8 mmol/l and 11.1 mmol/l • some patients with IGT develop diabetes mellitus later • Pregnancy IGT is treated as GDM because of the risks to the fetus.

33. Impaired fasting glucose (IFG) • Like IGT, refers to a metabolic stage intermediate between normal glucose homeostasis and DM. • Fasting venous plasma glucose ≥ 6.1 mmol/l but < 7.8 mmol/l 2h after an OGTT

34. Insulin resistance syndrome or metabolic syndrome • There is an aggregation of lipid and non-lipid risk factors of metabolic origin. • A particular cluster is Known metabolic syndrome, syndrome X or Reaven’s syndrome and is closely linked to insulin resistance. • Defined by the presence of three or more of the following features: • Abnormal obesity (west circumference): • Male > 102 cm (40 in) • Female > 88 cm (35 in)

35. Insulin resistance syndrome or metabolic syndrome • Fasting plasma TG > 1.7 mmol/l • Fasting plasma HDL-cholesterol: • Male < 1.0 mmol/l • Female < 1.3 mmol/l • blood pressure ≥ to 130/85 mm Hg • Fasting blood glucose > 5.5 mmol/l • Other associated features may include PCOS, fatty liver, raised fibrinogen and plasminogen activator inhibitor 1 concentrations, renal sodium retention, hyperuricaemia and dense LDL particles

36. Metabolic features of DM • Hyperglycemia • If plasma glucose > 10.0 mmol/l, glycosuria would be expected • High urinary glucose concentration produce osmotic diuresis and therefore polyuria. • Cerebral cellular dehydration due to hyperosmolality, secondary to hyperglycemia, causes polydepsia • Prolonged osmotic diuresis may cause excessive urinary electrolyte loss.

37. Metabolic features of DM • Abnormal in lipid metabolism These may be secondary to insulin deficiency • Lipolysis is enhanced and plasma NEFA concentration rise. • In the liver NEFA converted to acetyl CoA and ketones or re-esterified to form TG and incorporated into VLDL which accumulate in plasma because lipoprotein lipase, which require insulin for optimal activity to catabolise VLDL • HDL-cholesterol tend to be low in type 2 DM

38. Metabolic features of DM • Chylomicronaemia may also occur if insulin deficiency is very sever • Cholesterol synthesis increased with an associated increase in LDL • as a consequent, patients with DM may show high TG, raised cholesterol and low HDL-cholesterol

39. Long-term effects of DM Vascular disease is a common complication of DM • Macrovascular disease due to abnormalities of large vessels (coronary artery, cerebrovascular or peripheral vascular insufficiency), the condition probably related to alteration of lipid metabolism and associated hypertension. • Microvascular disease due to abnormalities of small blood vessels particularly affects the retina (diabetic retinopathy) and the kidney (nephropathy), both may be related to inadequate glucose control.

40. Long-term effects of DM Characteristic lesion: The renal complications may be partly due to the increase glycation of structural proteins in the arterial walls supplying the glomerular basement membrane; similar vascular changes in the retina may account for the incidence of diabetic retinopathy. • Infections are also more common in diabetic patients, e.g. urinary tract or chest infections • Diabetic neuropathy can occur • Diabetic ulcer, e.g. of the feet, can lead gangrene and amputation. • The joints can also affected • Skin disorder; e.g. abscesses

41. Monitoring of DM • Glycosuria • Defined as a concentration of urinary glucose detectable. • Usually, the proximal tubular cells reabsorb most of the glucose in the glomerular filtrate. • Glycosuria occur when the plasma, and therefore glomerular filtrate, concentrations exceed the tubular reabsorptive capacity. • When plasma glucose concentration > 10.0 mmol/l

42. Monitoring of DM • Blood glucose • Glycatedhaemoglobin HbA1c is formed by non-enzymatic glycation of haemoglobin and is dependent on: 1- mean plasma glucose concentration 2- lifespan of the red cell Falsely low values may be found in patients with haemolytic disease. HbA1c gives a retrospective assessment of the mean plasma glucose concentration during the preceding 6-8 weeks

43. Monitoring of DM The higher HbA1c, the poorer the mean diabetic or glycaemic control. • Fructosamine Plasma fructosamine may be used to assess glucose control over a shorter time course than that of HbA1c (about 2-4 weeks). Fructosamine reflect glucose bound to albumin, which has a plasma half-life of about 20 days but is problematic in patients with hypoalbuminaemia.

44. Monitoring of DM • Blood ketones • Urinary albumin determination and diabetic nephropathy One of the earliest signs of diabetic renal dysfunction is the development of small amounts of albumin in the urine (microalbuminuria). Normoalbuminuria < 30 mg/day or < 20 µg/min. Microalbuminuria 30-300 mg/day or 20-200 µg/min. Untreated microalbuminuria can progress to albuminuria (> 300 mg/day), impaired renal function and finally end-stage renal failure.

45. Monitoring of DM A random urine sample or timed overnight collection can be useful to assess urinary albumin excretion, although the standard test is the urinary albumin to creatinine ratio (ACR), which avoids a timed urine collection. ACR < 2.5 g/mol for male ACR < 3.5 g/mol for female

46. Acute metabolic complication of DM • Hypoglycemia • This is probably the most common cause of coma seen in Diabetic patients • Hypoglycemia is most commonly caused by overadministration of insulin plus inadequate food intake, increased exercise. • Overadministration of hypoglycemic medicine

47. Acute metabolic complication of DM • Diabetic ketoacidosis Insulin insufficiency triggers the following: • Increase lipid and protein breakdown • Enhanced hepatic gluconeogenesis and impaired glucose entry into cells The clinical consequence of diabetic ketoacidosis are due to: • Hyperglycemia causing plasma hyperosmolality • Metabolic acidosis • glycosuria

48. Acute metabolic complication of DM • Diabetic ketoacidosis • Vomiting water & electrolyte loos (increase fluid depletion)(extracellular depletion) shift of water out of the cellular compartment cellular dehydration • Haemoconcentration & glomerular filtration rate cause uraemia due to renal circulatory insufficiency • The rate of hydrogen ions production exceeds the rate of bicarbonate generation, which buffer H+, lead to plasma bicarbonate falls. H+ secretion causes a fall in urinary pH.

49. Acute metabolic complication of DM • Diabetic ketoacidosis 4. Failure of glucose entry into cells & glomerular filtration rate may raise plasma potassium concentration, before treatment is started.

50. Acute metabolic complication of DM • Hyperosmolal non-ketotic coma (HONK) (hyperosmolar hyperglycaemic state (HHS)) • Insulin is present to some degree :it suppress fat breakdown lack of ketosis • insulin is present to some degree : its effective is less than needed for effective glucose transport hyperglycemia glycosuria & polyuria body fluid depletion intracellular dehydration, which contribute to coma • Hypernatraemia due to predominant water loss is more commonly found than in ketoacidosis.