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Carbohydrates

Carbohydrates. Monosaccharides. D-glucose. D-galactose. Reducing substance : hydroxyl group near an aldehyde or ketone group can react with Cu 2+ , converting it to Cu +. D-glucose. D-fructose. Disaccharides. reducing end. Lactose. reducing end. Maltose. Sucrose. not a reducing

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Carbohydrates

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  1. Carbohydrates • Monosaccharides D-glucose D-galactose

  2. Reducing substance: hydroxyl group near an aldehyde or ketone group can react with Cu2+, converting it to Cu+ D-glucose D-fructose

  3. Disaccharides reducing end Lactose

  4. reducing end Maltose

  5. Sucrose not a reducing substance

  6. Polysaccharides reducing end Cellulose

  7. Amylopectin Glycogen branches about every 24-30 linear linkages branches every 8-12 glucose units

  8. Intestinal absorption of carbohydrates microvilli Jejunum villi

  9. Intestinal absorption of carbohydrates MICROVILLI BRUSH BORDER GUT BLOOD 1 maltose a -amylase Starch saliva and pancreatic juice glucose Glycogen sucrose 2 fructose 3 lactose galactose galactose Monosaccharides glucose fructose

  10. Glucose Production Glucose Consumption 125g Blood glucose brain Glucose 50g Glycogen (75%) 50g rbc wbc muscle fat cell

  11. Glucose transporters *insulin low…GLUT4 in intracellular compartments; insulin high…GLUT4 translocates to membrane

  12. Glucose Production Glucose Consumption CO2 125g Blood glucose brain Glucose 50g Glycogen (75%) 50g rbc wbc pyruvate lactate (10-15%) certain amino acids (10-15%) CO2 glycerol (2%) muscle fat cell

  13. Regulation of blood glucose • Glycogenesis: glucose glycogen (liver, muscle) • Glycogenolysis: glycogen glucose • Gluconeogenosis: non-CHO sources glucose • Glycolysis: glucose CO2 + H2O + ATP • Renal threshold: proximal convoluted tubule

  14. Regulation of blood glucose stimulates Somatostatin inhibits d Pancreatic Islet a b Cortisol Growth hormone glucose glucagon Insulin epinephrine Glycogenolysis Gluconeogenesis Glucose uptake Glycolysis Glucose uptake Lipogenesis Muscle Liver Adipose tissue

  15. Determination of glucose • Specimens used • whole blood • used with home glucose monitoring units • cellular use of glucose gives 7% decrease/hour • NaF preserves glucose 24hr, RT • cannot use such a specimen for enzyme assays, especially urease • lithium iodoacetate preserves glucose; does not interfere with urease • capillary blood = fasting venous level + 5 mg/dL • plasma, serum • 10-15% higher level than whole blood glucose • RI: 70-105 mg/dL • CSF • RI: 60-70% plasma glucose = 40-70 mg/dL • urine • RI: <30 mg/dL random; <500 mg/24 hr

  16. Glucose Methods HK • hexokinase glucose + ATP gluc-6-PO4 + ADP gluc-6-PO4 + NAD+ 6-phosphogluconate + NADH + H+ INT + NADH + H+formazan + NAD+ • most widely used • reference method against which others are compared • serum, plasma and urine • avoid hemolysis G6PD PMS

  17. Glucose Methods • glucose oxidase • glucose + O2 gluconic acid + H2O2 • H2O2 + reduced dye oxidized dye+ H2O • peroxidase reaction interference by uric acid, vitamin C, bilirubin • suitable for spinal fluid • measure O2 consumption via pO2 electrode • suitable for all body fluids • measure H2O2 production via H2O2 electrode • suitable for plasma, serum, whole blood GO POD

  18. Glucose Methods GDH • glucose dehydrogenase glucose + NAD+ D-gluconolactone + NADH + H+ • highly specific as “GDH-NAD”, with little interference, EXCEPT… • Pyrroloquinolinequinone (“GDH-PQQ”) • 2005 FDA warning • giving false increased glucose readings when patient is receiving maltose, icodextrin (dialysis), galactose, d-xylose

  19. Glucose Methods GDH • glucose dehydrogenase glucose + NAD+ D-gluconolactone + NADH + H+ • highly specific as “GDH-NAD”, with little interference, EXCEPT… • Pyrroloquinolinequinone (“GDH-PQQ”) • 2005 FDA warning • giving false increased glucose readings when patient is receiving maltose, icodextrin (dialysis), galactose, d-xylose • Coulometry • http://www.medisense.com/au • FreeStyle glucometer, Abbott Laboratories • Electrons released in the reaction are measured as a current • Allows very small volume (0.3 uL) to be used, with results in 15 seconds

  20. Glucose Methods • oxidation-reduction reactions Fe3+Fe2+ or Cu2+Cu1+ • least specific for glucose

  21. Clinical Significance • Hyperglycemia • diabetes mellitus • endocrine disorders • acromegaly: incr. growth hormone • Cushing’s syndrome: incr. cortisol • thyrotoxicosis: incr. T4 • pheochromocytoma: incr. epinephrine • drugs • certain anesthetics • steroids

  22. Clinical Significance • Hypoglycemia • insulin overdose • drugs • sulfonylureas • antihistamines • alcoholism (long term) • insulinoma • galactosemia • glycogen storage diseases

  23. Expert Committee on the Diagnosis and Classification of DM - 2005 • Diagnosis of Diabetes Mellitus • Symptoms of diabetes mellitus • Polyuria • Polydipsia • Unexplained weight loss • Any TWO of the following tests, on different days • Casual plasma glucose > 200 mg/dL • Fasting plasma glucose (FPG) > 126 mg/dL • 2hr Post prandial glucose (PPG) > 200 mg/dL after a meal with 75g glucose load

  24. Expert Committee on the Diagnosis and Classification of DM - 2005 • Type 1 • Type 1a • characterized by beta cell destruction caused by an autoimmune process, usually leading to absolute insulin deficiency • patients must take insulin to survive • usually young, with acute onset (days to weeks) • islet-cell antibodies usually present • Type 1b • idiopathic

  25. Expert Committee on the Diagnosis and Classification of DM - 2005 • Type 2 • insulin resistance in peripheral tissue and an insulin secretory defect of the beta cell • variable [insulin] • highly associated with a family history of diabetes, older age (>40), obesity and lack of exercise • more common in • Women • African American • Hispanics • Native Americans

  26. Expert Committee on the Diagnosis and Classification of DM - 2005 • “Other specific types” • pancreatic, hormonal disease • Pancreatitis, cystic fibrosis • Acromegaly (GH), Cushing’s syndrome (cortisol) • drug/chemical toxicity • insulin receptor abnormalities • no renal or retinal complications

  27. Expert Committee on the Diagnosis and Classification of DM - 2005 • Gestational diabetes mellitus • pregnancy • frequent but transitory glucose intolerance • greater risk of perinatal complications • placental lactogen? • > 140 mg/dL one hour after 50-g glucose load screening • TWO of four results abnormal in 100 g glucose load test: • fasting plasma glucose > 105 mg/dL • > 195 mg/dL at 1 hr • > 165 mg/dL at 2 hrs • > 145 mg/dL at 3 hrs

  28. National Diabetes Association 2003 • Pre-diabetes • http://diabetes.niddk.nih.gov/dm/pubs/diagnosis/index.htm • http://www.diabetes.org/pre-diabetes.jsp • Fasting Plasma Glucose Diagnosis Result (mg/dL) • 70 – 99 Normal • 100 to 125 Pre-diabetes (impaired fasting glucose) • 126 and above Diabetes mellitus* • *Confirmed by repeating the test on a different day.

  29. Clinical Significance • Glucose tolerance test (still used for gestational diabetes diagnosis) • patient preparation • normal diet three days prior to test • no food after regular evening meal on day before test • take fasting blood, urine specimen • drink 100 g glucose load within 5 minutes • allow water, but no food, chewing gum, smoking, exercise during test • specimens taken 1, 2, 3 hours after ingestion 200 Plasma glucose (mg/dL) Diabetic Normal 100 60 120 180 Minutes after glucose ingestion

  30. Clinical Significance • Other conditions, tests associated with diabetes mellitus • white cell antigens • HLA types DR3, DR4, DQB1*0302 • Note that diabetes resistance genes = DR2, DQB1*0602 • lipid studies • hyperlipoproteinemia type IV • increased TG • microalbuminuria • microangiopathies • retinal, renal, neural

  31. Management of diabetes mellitus • Glycated hemoglobin (A1) COOH COOH COOH COOH H2N H2N H2N H2N COOH COOH H2N H2N N N glu glu non-enzymatic process conversion of HbA into HbA1 at N-terminal valine

  32. Management of diabetes mellitus • Glycated hemoglobin • irreversible reaction occurring throughout the 120-day life span of rbc • reflects timed average [glucose] over previous 4-8 weeks • HbA1c = 80% total glycohemoglobin • reference range: 3-6% total Hgb • uncontrolled diabetes mellitus: 12-20% total Hgb • controlled: 9-12% total Hgb • Considerations when measuring HbA1c • abnormal hemoglobins can also be glycated • variability in levels of “labile fraction” (intermediates)

  33. Management of diabetes mellitus For every 1% decrease in HbA1c, risk of microvascular complications is reduced by 35% Diabetes Care 2000, 23: S27-S31

  34. Management of diabetes mellitus • Manual Methods for HbA1c compared Ion-exchange chromatography Affinity Chromatography + HbA--Val--N | CH2 | C=O | HOCH | HCOH | HCOH | CH2OH Principle + + + + HbA HbA1c + + + + pba -- - - - - - - - - - - - - - - -- -- - - pba pba “Fast fraction” HbA1c elutes first HbA1c elutes last pba Phenylboronic acid

  35. Management of diabetes mellitus • Manual Methods for HbA1c compared • other non-glycated Hb measured • IEC: HbF and any others with charge like A1 • AC: none • time • IEC: 2-3 hrs • AC: 15 minutes • glycated hemoglobins measured • IEC: A1a, 1b, 1c only • AC: any glycated hemoglobin, including abnormals • temperature sensitive? • IEC: yes • AC: no

  36. Management of diabetes mellitus • Automated Method for HbA1c • High pressure liquid chromatography (HPLC) • Cation exchange method • the eluant must be __________________ • The first form of hemoglobin eluting from the column must be _________________________ • The last form of hemoglobin eluting from the column must be __________________________

  37. Management of diabetes mellitus • Attempts to convert HbA1c value to “mean blood glucose” value • Nathan et al, 1984 using linear regression on data from 21 patients • 33.3 (%HbA1c) – 86 • Examples: 6.0% = ~115 mg/dL 7.5% = ~165 mg/dL 9.0% = ~215 mg/dL

  38. Management of diabetes mellitus • Revised calculation, effective 3/21/05 • Rohlfing et al, 2002 using comparison data from 1500 patients • (35.6 x %HbA1c) – 77.3 • Examples: 6.0% = ~136 mg/dL 7.5% = ~190 mg/dL 12.0% = ~350 mg/dL • Only valid for A1c values between 6 and 12%

  39. Management of diabetes mellitus • Glycated serum proteins • albumin (“fructoseamine”) • turnover = 2-3 weeks • rapid method using tetrazolium dye reduction colored product

  40. Carbohydrate inborn errors of metabolism • Glycogen storage diseases • lack of enzymes of glycogen metabolism • incr. tissue glycogen • results in severely limited lifespan • von Gierke’s disease • liver cells lack glucose-6-phosphatase glucose-6-PO4 blood glucose triose phosphate pyruvate

  41. Carbohydrate inborn errors of metabolism • Lactose intolerance • deficiency in intestinal mucosal lactase • GTT done as baseline • 2nd day, give lactose instead of glucose • normal: normal GTT curve • abnormal: flat curve ( and much pain!)

  42. Carbohydrate inborn errors of metabolism • Galactosemia (1) galactose galactose-1-PO4 galactilol (2) galactose-1-PO4 UDP-galactose (3) UDP-galactose UDP-glucose cataracts (4) UDP-glucose glucose-1-PO4 • deficiency in uridyl transferase * • results in galactosuria, retardation, cataracts, no conjugation of bilirubin • urine tests * gal. oxidase galactose + O2 galactose dialdehyde + H2O2

  43. Ketones • Complication of uncontrolled diabetes mellitus • Acid-base imbalance • Can be life-threatening • Acetone, acetoacetate, b-hydroxybutyrate Blood ketones amino acids acetyl CoA fatty acids Ketones acetoacetate B-hydroxybutyrate acetate CO2 TCA cycle H2O ATP

  44. Ketones • Complication of uncontrolled diabetes mellitus • Sodium nitroprusside • B-hydroxybutyrate dehydrogenase Blood ketones amino acids acetyl CoA fatty acids Ketones acetoacetate B-hydroxybutyrate acetate CO2 TCA cycle H2O ATP

  45. Extra slides

  46. Glycogenesis, glycogenolysis • Hormones involved • fed: insulin from pancreatic beta cells (Islets of Langerhans) • preproinsulin proinsulin (A, B and C peptides) insulin + C-peptide • anabolic (synthesis) • promotes cellular uptake of glucose • increased: • lipogenesis • protein synthesis • glycogenesis • decreased: • lipolysis • ketone formation • gluconeogenesis • glycogenolysis

  47. Glycogenesis, glycogenolysis • Hormones involved • fasting: glucagon from pancreatic alpha cells • catabolic • liver: glycogen converted to glucose, released into blood • muscle: glycogen converted to glucose-6-PO4, remains in the muscle cell for its own energy needs • “fight or flight” : epinephrine from adrenal medulla • action similar to glucagon

  48. Glycogenesis, glycogenolysis • Stimulation of insulin release • glucose • leucine, arginine, histidine, phenylalanine • sulfonylureas (tolbutamides) • ACTH, GH • Inhibition of insulin release • thiazide diuretics • dilantin (antiseizure) • human placental lactogen (diabetes of pregnancy) • Decreased tissue response to insulin • glucocorticoids obesity • estrogens inactivity • progestins low CHO diet

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