Transcript cl biochem 4-carbohydrate metabolism

MGV - CLINICAL BIOCHEMISTRY
CARBOHYDRATE
METABOLISM
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
1.
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
2. Glucagon:
• Secretion stimulated by hypoglycemia,
gluconeogenic aminoacids and inhibited
by glucose, insulin, somatostatin
• Stimulates glycogenolysis,
gluconeogenesis, rising the glycemia
HORMONAL CONTROL
3. Growth hormone
– Secretion stimulated by hypoglycemia
– Action: incresed glucose production in the liver, reduced
uptake by some tissues
4. Adrenaline
– Secretion stimulated by hypoglycemia
– Action: glycogenolysis, reduces insulin secretion resulting
increasing the glucose concentration
5. 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 hormonesensitive 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 phydroxybenzene 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.
1. Lactose: exists physiologically in late pregnancy and lactation.
2. Galactose: in infants during lactation;galactosemia (associated with
hypoglycemia);
3. Fructose: after fruit ingestion, pregnancy, lactation; fructose
intolerance, essential fructosuria.
4. Pentose: chronic pentosuria (deficiency of the metabolism of
glucogenetic amino acids).
GLUCOSE IN THE URINE
.
When other saccharides are present, they need to be identified.
1. Lactose: exists physiologically in late pregnancy and lactation.
2. Galactose: in infants during lactation;galactosemia (associated with
hypoglycemia);
3. Fructose: after fruit ingestion, pregnancy, lactation; fructose
intolerance, essential fructosuria.
4. 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%