Transcript Immunoturbidimetric assay

CORE AREA 1
- CHRONIC DIABETES CASE E
Mrs HT, 60 years, has
essential hypertension &
NIDDM.
Regular Medications Taken
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Felodipine
Atenolol
Chlorothiazide
Metformin
10mgs BD
50mgs mane
500mgs mane
500mgs TDS
A recent 24 hour urine shows:
Urine albumin 50 mgs
(Normal range is less than 15 mgs in 24
hours)
Discussion:
What is the relevance of the
clinical chemistry result for the
diabetic patient especially with
respect to the possibility of long
term complications.
RELEVANCE
• Poor control of diabetes is associated with
enlargement of the kidneys & initially a
high GFR, which are early functional
abnormalities in diabetic nephropathy.
• Microalbinuria is a marker of renal damage
in the diabetic state.
• Patients with microalbinuria over time
develop progressive renal injury.
Microalbinuria
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A marker of the development of proteinuria
Early mortality in NIDDM patients
Associated with unfavorable lipid profiles
Increasing blood pressure elevation
Retinopathy
Untreated it is progressive
Progressive decline in GFR
Higher level of structural damage present
Microalbinuria
• Poor metabolic control is a risk factor for
the development and progression of
microalbinuria as well as a decline in GFR
• Diabetic nephropathy progresses over 10 to
25 years, it is the most common cause of
end stage renal disease in the USA
• Diabetic nephropathy ultimately presents as
the nephrotic syndrome
The Nephrotic Syndrome
• Results from alterations in the
permselective characteristics of the
Glomerular Basement Membrane (caused
by hyperglyceamia), that allows increased
passage of proteins of intermediate size into
the urine
The Nephrotic Syndrome
• Consists of heavy proteinuria,
hypoalbuminemia, hyperlipidemia, higher
concentration of high MW proteins &
oedema formation, associated with
hypertension.
Methods used to measure
urine albumin level.
Methods to measure urine albumin
level
Quantitative assay
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Radioimmunoassay
Immuno-nephelometry
Electro-immuno-diffusion assay
Immunoturbidimetric assay
Semi-quantitative assay
- test strip
Methods to measure urine albumin
level
*Radioimmunoassay
-Sensitive,precise and rapid method
-Reliable results
*Immuno-nephelometry and Electro-immunodiffusion assay
-sensitive and accurate
-correlated with RIA
Methods to measure urine albumin
level
*Immunoturbidimetric assay
-for initial screening of albuminuria
*Semi-quantitative method
-test strip
-high sensitivity but poor specificity
-use of this method questionable
Drugs that can affect urine
albumin level
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Acetazolamide
Aminoglycosides
Amphotericin
Cephalosporins
Colistin
Griseofulvin
Lithium
Nephrotoxic drugs eg arsenicals, gold salt
Penicillamine
Drugs that can affect urine
albumin level
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Penicillin G
Polymyxin B
Salicylates
Sulfonamides
Tolbutamide
Disease factors that could affect
urine albumin level
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Bacterial pyelonephritis
Bladder tumor
Congestive heart failure
Diabetic nephropathy
Glomerulonephritis
Heavy metal poisoning
Lupus erythematosus
Disease factors that could affect
urine albumin level
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Malignant hypertension
Multiple myeloma
Nephrotic syndrome
Nephrotoxic drug therapy
Polycystic kidney disease
Preeclampsia
Urinary tract infection
Other interfering factors for urine
albumin
• Severe emotional stress
• Strenuous exercise
• Urine contamination with secretion
Advanced Glycation Endproducts
(AGE)
What Are AGEs?
• A class of complex products, brown in colour
and with specific fluorescence
• Very unstable, reactive compounds and hence
difficult to analyse completely
• The result of a succession of chemical
reactions linked in a complex network (known
as the Maillard reaction)
• Binding of AGEs to RAGE (AGE receptors)
activates endothelial cells and monocytes to
produce cytokines, adhesion molecules and
tissue factors.
AGE Production
• Non-enzymatic glycosylation between a free amino group
on a protein and a carboxyl group from a sugar e.g.
glucose to form Schiff’s base
• Schiff base formation is relatively fast and highly
reversible and is an equilibrium reaction. (The amount of
Schiff base formed is dictated by glucose concentration.)
• Over 24-48 hours Schiff’s base undergoes molecular
rearrangement to create an N-substituted glycosylamine
• This leads to formation of more stable Amadori products
(1-amino-1-deoxy-2-ketose)
How are they formed? Continued….
• The formation of the Amadori product from the
Schiff base is slower, but much faster than the
reverse reaction and therefore tends to accumulate
on proteins. (Equilibrium levels of Amadori
products are reached in weeks)
• As with the formation of the Schiff base, the
amount of Amadori product formed is related to
the glucose concentration.
• On long lived proteins Amadori products
rearrange further to form AGEs
The formation of advanced
glycosylation end-products (AGE).
• Best chemically characterised AGE
compounds found in humans are pentosidine
and carboxyl methyl lysine.
• AGES may also originate from arabinose and
xylose from food, e.g., intestinal bacteria
degrade xylanes found in fruit to produce
AGEs
• AGEs are toxic, inducing bacterial
mutagenesis
• Formed in excess in diabetes mellitus, renal
failure and the elderly.
AGE and RAGE (Receptor for AGE)
• RAGE receptors are expressed on a wide
range of cells including smooth muscle
cells, monocytes, macrophages, endothelial
cells and astrocytes.
• The RAGE receptor is thought to mediate a
complex pattern of intracellular signalling.
• RAGE expression is increased in the blood
vessels and kidneys of diabetic patients.
What effect do AGEs have on the
body?
Relevance to Diabetes.
• AGEs are believed to contribute to the
chronic complications (Secondary
complications) of diabetes mellitus.
• Which include: Visual Impairment,
Blindness (Retinopathy), Renal Failure
(Nephropathy), Stroke, MI, Neuropathy and
other Cardiovascular Complications.
Relevance to Diabetes.
(Nephropathy)
• NEJM conducted a study to measure serum
and tissue AGEs in diabetic (Types I and
Type II) and nondiabetic patients with
different levels of renal function.
• The mean AGE content of samples of
arterial-wall collagen from 9 diabetic
patients was significantly higher than that of
samples from 18 nondiabetic patients.
Relevance to Diabetes.
(Nephropathy) Continued …..
• In diabetic patients: The levels of AGE peptides
correlated directly with serum creatinine and
inversely with creatinine clearance, suggesting
that levels of AGE peptides increased with the
severity of diabetic nephropathy.
• Conclusion: AGEs accumulate at a faster-thannormal rate in arteries and the circulation of
patients with diabetes; the increase in circulating
AGE peptides parallels the severity of renal
functional impairment in diabetic nephropathy
Relevance to Diabetes (Vascular)
• Initial AGE- RAGE interaction- cellular
activation and inflammation.
• Followed by lipoprotein accumulation and
atherosclerosis.
• AGE also depletes nitric oxide.
• Resulting in vascular thickening with loss
of elasticity and hypertension.
Relevance to Diabetes (Vascular)
Continued….
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Schematic description of the formation of collagen cross-links. AGEs
can react with free amino groups on an adjacent protein to form
cross-links. Cross-linking can lead to decreased compliance of large
vessels and of the myocardium
Relevance to Diabetes
(Retinopathy)
• Characterised by abnormal vessel
development leading to haemorrhages.
• Proposed that AGE may increase retinal
endothelial call permeability resulting in
vascular leakage.
Conclusion.
• Measuring the concentration of serum
AGE’s is becoming more important as
studies are concluding that they are useful
as a marker for or indication of possible
secondary diabetic complications.
Haemoglobin A1C. (Also known as
Glycosylated Hb)
• An amadori glycoylation product.
• Is the stable product of nonenzymatic glycosylation of the
B- chain of Hb by plasma glucose.
• Indicator of plasma glucose control over the previous 1-3
months.
• Normal levels is around 6%.
• In poorly controlled diabetics level ranges from 9- 12%.
• HbA1c is not a specific test for diabetes but often indicates
existing diabetes.
• Often viewed as a measure of long term control.
Fructosamine
• Because the turnover rate of proteins is much faster than that of
hemoglobins, the measurement of glycated serum proteins (i.e.,
fructosamines) provides a shorter term, more rapidly responding
assessment of diabetic control. The fructosamine measurement reflects
an average blood glucose level of the past 2-3 weeks, while GHb
values indicate control over a 2-3 month period.
Fructosamine levels are directly proportional to serum protein levels
(albumin, immunoglobulins). Since the fructosamine level is not given
as a percentage of actual serum total protein, a low level may indicate
good control or it may indicate low total protein.
Variations in levels of serum proteins (albumin, IgA, and other
immunoglobulins) may affect fructosamine results.
• Non-diabetic patient: 0-285 µmol/L
References
Australian Medicines Handbook 2002 – Simone Rossi, Agnes
Vitry, Eve Hurley, Fay Abbott, Sharon Goldsworthy, Christopher
Alderman, Manya Angley, Neil Hotham. Finsbury Press 2002.
Diseases of the Kidney 6th Edition Volume 1 – Robert W
Schrier, Carl W Gottschalk. Little, Brown and Company 1997.
Nephrology Volume 1 – Jean Hamburger, G Richet, J Crosnier,
J L Funck-Brentano, B Antoine, H Ducroit, J P Mery and H de
Montera. W. B. Saunders Company 1968.
Papper’s Clinical Nephrology 3rd Edition – Francisco Llach.
Little, Brown and Company 1993.
Renal Pathology 2nd Edition – Frederick Dische. Oxford
Medical Publications 1995.
The Merck Manual 17th Edition – Mark H Beers, Robert
Berkow. Merck Research Laboratories 1999.
Therapeutic Guidelines Antibiotics Version 11 – J Spicer.
Therapeutic Guidelines Limited 2002.