URINARY ALBUMIN EXCRETION (UAE) : INTRODUCTION AND ITS IMPLICATIONS

Patients with diabetes mellitus are at high risk of suffering renal damage. Diabetes is the most common cause of end -stage renal disease (ESRD). Although nephropathy is less common in patients with type 2 diabetes, approximately 60% of all cases of diabetic nephropathy occur in these patients because of the higher incidence of this form of diabetes. Early detection of diabetic nephropathy relies on tests of urinary excretion of albumin. Persistent proteinuria detectable by routine screening tests (equivalent to a urinary albumin excretion [UAE] rate greater than or equal to 30 mg/d) indicates overt diabetic nephropathy. Once diabetic nephropathy occurs, renal function deteriorates rapidly and renal insufficiency evolves. Treatment at this stage can retard the rate of progression but not stop or reverse the renal damage. Preceding this stage is a period of increased UAE not detected by routine methods. This range of 20 to 200 μg/min (or 30 to 300mg/24hr or albumin/creatinine ratio of 30-300 μg/mg) of increased UAE defines microalbuminuria. Note that it is not defined in terms of urinary albumin concentration, although the albumin: creatinine ratio can be used as a substitute for albumin measurements in a time collection of urine. The term microalbuminuria implies a small version of the albumin molecule rather than an excretion rate of albumin greater than normal but less than that detectable by routine methods. Clinical proteinuria or microalbuminuria is established with an albumin-creatinine ratio of ≥300 μg/mg or protein excretion ≥300 mg/day. 

The presence of increased UAE denotes an increase in the transcapillary escape rate of albumin and is therefore a marker of microvascular disease. Persistent UAE greater than 30 mg/d represents a twentyfold greater risk for the development of clinically overt renal disease in patients with type 1 and type 2 diabetes. Prospective studies have demonstrated that increased UAE precedes and is highly predictive of diabetic nephropathy, end-stage renal disease, cardiovascular mortality, and total mortality in patients with diabetes mellitus. In addition increase UAE identifies a group of nondiabetic subject at increased risk of coronary artery disease.

UAE is increased by physiological factors (e.g., exercise, posture, and diuresis) and the method of urine collection must be standardized. Samples should not be collected after exertion, in the presence of urinary tract infection, during acute illness, immediately after surgery, or after an acute fluid load. All the following urine samples are currently acceptable: 

(1) 24-hour collection; 

(2) overnight (8 to 12 hours, timed) collection; 

(3) 1- to 2-hour timed collection (in laboratory or Clinic); or 

(4) first morning sample for simultaneous albumin and creatinine measurement. 

Only results for timed specimens can be reported as mg albumin excreted per hour, but the albumin: creatinine ratio is more practical and convenient for the patient and is the recommended method. A first morning void sample is best because it has a lower within-person variation for the albumin: creatinine ratio than a random urine sample. At least three separate specimens, collected on different days, should be assayed because of the high intraindividual variation diurnal variation (50% to 100% higher during the day). Urine should be stored at 4⁰C after collection. Alternatively, 2 mL of 50 gm/L sodium azide can be added per 500 mL of urine, but preservatives are not recommended for some assays. Bacterial contamination and glucose have no effect. Specimens are stable for 2 weeks at 4 'C and for at least 5 months at -80⁰C. Albumin concentration decreased by 0.27% at -20⁰C. Freezing samples has been reported to decrease albumin, but mixing immediately before assay eliminates this effect.

The test strips most of which are optimized to read positive at predetermined albumin concentration have been recommended for screening programs. Test strips contains bromophenol blue in alkaline matrix to detect albumin concentrations exceeding 40 mg/L. Other test strips include antialbumin IgG complexed to galactosidase. The albumin in the urine binds to antibody enzyme conjugate in the test strip. Excess conjugate is retained in a separate zone containing immobilized albumin and only albumin bound to the antibody-enzyme immunocomplex diffuses to the reaction zone. Here it reacts with a buffered substrate (chlororphenol red galactoside) to produce a red color when the beta galactosidase hydrolyzes galactose.

For quantitation different RIA, ELISA radial immunodiffusion and immunoturbidimetry are available.

The ADA recommends initial UAE measurement in type 1 diabetes patients who have had diabetes more than or equal to 5 years and in all type 2 diabetic patients. Because of the difficulty in dating the onset of type 2 diabetes, screening should commence at diagnosis. Analysis should be performed annually in all patients who have a negative screening results. If screening result is positive UAE should be evaluated by quantitative assay. Diagnosis requires the demonstration of increased UAE in at least two of 3 tests measured within 6 month period.

(Source: Tietz Clinical Chemistry, 4th Edition)

TEST FOR INSULIN RESISTANCE

Subjects requiring large amount of insulin to maintain euglycaemia e.g. >150 units or 1.5 units/kg body weight/day, insulin resistance may be postulated. For this insulin is administered intravenously and subcutaneously and the level of glucose and insulin in plasma is measured. Normal fasting insulin concentration are up to 20 mU/L. Hyperinsulinaemic clamp is the reference measure of insulin resistance. In euglycaemic variant of the test, insulin is infused into a peripheral vein so as to raise the plasma insulin concentration to a target range around 60 mU/L. 

The plasma glucose concentration is measured every 5-10 min and glucose is infused peripherally to maintain glucose concentraions within the desired range. When a steady state has been reached (usually 90-120 min), the rate of exogenous glucose infusion needed to maintain the glucose concentration is an index of the glucose clearance rate and of the subject’s insulin sensitivity.

Glucose transporter function can be assayed by incubating cells of interest (e.g. leukocytes, monocytes, adipocytes) with a non-metabolizable glucose analog such as 2-deoxyglucose. The cellular content of the glucose analogue after a given time provides a measure of glucose transporter function.  

MEASUREMENT OF β- CELL FUNCTION

Measurement of plasma C-peptide concentration can be done. Elevated fasting plasma proinsulin indicates subjects with abnormal beta cell function, even if glucose tolerance is normal. 

ADVANCED GLYCATION END PRODUCTS: AN INTRODUCTION

The molecular mechanism by which hyperglycemia produces toxic effect is unknown, but glycation of tissue proteins may be important. Nonenzymatic attachment of glucose to long lived proteins like collagen or DNA, produces stable Amadori early Glycated products. These undergo a series of additional rearrangements dehydration and fragmentation reactions, resulting in stable advanced glycation end products (AGE). The amounts of these products do not return to normal when hyperglycemia is corrected and they accumulate continuously over the lifespan of the protein. Hyperglycemia accelerates the formation of protein-bound AGE, and patients with diabetes mellitus thus have more AGE than healthy subjects. Through effects on the functional properties of protein and extracellular matrix, AGE may contribute to the microvascular and macrovascular complications of diabetes mellitus. Moreover an inhibitor of AGE formation, aminoguanidine has been shown to prevent several complications of diabetes in animal model.

In healthy people Hb-AGE accounts for 0.4% of circulating Hb, with significantly higher in diabetes mellitus. After acute change in glycemia, Hb-AGE level changes, but the rate of alteration is 23% slower than that of HbA_1c. Thus Hb-AGE provides a measure of diabetic control longer than that indicated by GHb, reflecting blood glucose concentration over a greater proportion of life of red blood cells. 

Fructosamine and it's implications

FRUCTOSAMINE

Fructosamine is a ketoamine product of protein glycation formed when glucose bound to variety of proteins by aldimine linkage undergoes an Amadori rearrangement. The major component of fructosamine in plasma is Glycated albumin. Fructosamine is easily measured (using nitroblue tetrazolium assay); its concentration reflects control over the preceding 15-20 days. When the patient has abnormal hemoglobins, or during pregnancy alternative tests should be used. Glycated albumin and Glycated fibrinogen are proposed for such conditions. Albumin has half life of approximately 20 days, so fraction that is Glycated reflects glycaemic control for the preceding 1-2 weeks.

(Source: Tietz Clinical Chemistry, 4th Edition)

Fructosamine is the generic name for plasma protein ketoamine. There is interaction of glucose with the ε-amino group on lysine residue of albumin. Because all Glycated serum proteins are fuctosamines and albumin is the most abundant serum protein, measurement of fructosamine is thought to be largely a measure of Glycated albumin. As fructosamine determination monitors short term glycemic changes different from GHb, it may have a role in conjunction with GHb rather than instead of it. In addition fructosamine may be useful in patients with hemoglobin variants such as HbS or HbC that are associated with decreased erythrocyte lifespan where GHb is of little value. Fructosamine values are highly affected and not recommended in conditions that affect protein turnover like liver cirrhosis, nephrotic syndrome or dysproteinemias, inflammatory conditions. It is generally accepted that the test should not be performed when serum albumin is less than 30g/L.

 

Methods for measuring Glycated proteins include affinity chromatography using immobilized phenylboronic acid, HPLC of Glycated lysine residue after hydrolysis of Glycated proteins, photometric procedure in which mild acid hydrolysis releases 5-hydroxymethylfurfural- proteins are precipitated with TCA and the supernatant is reacted with 2-thiobarbituric acid; and other procedures using phenylhydrazine and furosine.  Another method is under alkaline conditions which results in fructosamine undergoing an Amadori rearrangement and the resultant compounds having reducing activity that can be differentiated from other reducing substances. In the presence of carbonate buffer, fructosamine rearranges to the eneaminol form, which reduces NBT to a formazan. The absorbance at 530 nm is measured at two time points and the absorbance change is proportional to the fructosamine concentration. 

TEST FOR RECENT GLYCAEMIC CONTROL: HbA1c measurement

MEASUREMENT OF GLYCATED HEMOGLOBIN

Glycation is the non enzymatic addition of sugar residue to amino groups of proteins. In adults HbA constitute the major fraction (97%) also has other subforms namely A_1a, A_1b, A_1c which are collectively called HbA₁, fast hemoglobins, glycohemoglobins or Glycated hemoglobins. HbA_1c is formed by the condensation of glucose with N-terminal valine residue of each β-chain of HbA to form an unstable Schiff base (aldimine, pre-HbA_1c). The Schiff base may either dissociate or undergo an Amadori rearrangement to form a stable ketoamine, HbA_1c. HbA_{1a1, 1a2} which make up HbA_1a have fructose-1, 6-diphosphate and glucose-6-phosphate, respectively attached to amino terminal of the β-chain. Other are HbA_1b has pyruvate attached to N-terminal of beta chain.  HbA_1c is the major fraction constituting approximately 80% of HbA₁.

Glycation may also occur at sites other than the end of beta chain, such as lysine residue or the alpha chain. These GHbs referred to as Glycated HbA₀ or total Glycated Hb. These are measured by boronate affinity chromatography.

(Source: Tietz Clinical Chemistry, 4th Edition)

Formation of GHb is essentially irreversible and the concentration in the blood depends on both the lifespan of the red blood cell (average 120 days) and the blood glucose concentration. Since erythrocyte is free permeable to glucose. Because the rate of formation of GHb is directly proportional to the concentration of glucose in the blood, the GHb concentration represents the integrated values for glucose over the preceding 6 to 8 weeks. This provides an additional criterion for assessing glucose control because GHb values are free of day to day glucose fluctuations and are unaffected by recent exercise or food ingestion.

The interpretation of GHb depends on the red blood cells having a normal lifespan. Patients with hemolytic disease or other conditions with shortened red blood cells survival exhibit a substantial reduction in GHb. Similarly individuals with recent significant blood loss have false low values owing to higher fraction of young erythrocytes. High GHb concentrations have been reported in iron deficiency anemia, probably because of high proportion of old erythrocytes. Presence of other hemoglobinopathies can alter results. Presence of carbamylated Hb which is formed by attachment of urea and is present in large amount in renal failure and common in diabetic patients, also produce altered results.

GHb has been established as an index of long term blood glucose concentration and as a measure of the risk for the development of complications in patients with diabetes mellitus. There is direct relationship between blood glucose concentration (assessed by HbA_1c) and the risk of complications. The absolute risks of retinopathy and nephropathy were directly proportional to the mean HbA_1c. Studies have shown reduction in HbA_1c level will significantly reduce the risk of microvascular complications and retinopathy and nephropathy and cardiovascular disease. ADA recommends that a primary treatment goal in adults with diabetes should be near normal glycemia with HbA_1c <7%. HbA1c of 7% (of total HbA) corresponds with mean plasma glucose of approximately 170 mg/dl, and each 1% increase with a 36 mg/dl increase in mean plasma glucose concentrations.

There are more than 30 different methods for determination of GHbs. These methods separate hemoglobin from GHb using technique based on charge differences (ion-exchange chromatography, HPLC, electrophoresis, IEF), structural differences (affinity chromatography and immunoassay), or chemical analysis (photometry and spectrophotometry). The result in all is expressed as percentage of total Hb.

Ion exchange chromatography separates Hb variants on the basis of charge. The cation exchange resin (negatively charged) packed in disposable minicolumn has an affinity for Hb, which is positively charged. The patient’s sample is hemolyzed and an aliquot of the hemolysate is applied to the column. A buffer is applied and the eluent collected. Here GHb is less positively charged than other so will elute first than other. The eluted GHb (A1a, 1b and 1c, collectively A1) are measured in spectrophotometer. Other Hbs are also measured after subsequent elution and the HbA1 is expressed as percentage of total.

HPLC can be used for separation and quantitation of HbA1c and other fractions. HPLC employs, cation exchange chromatography.

Agar gel electrophoresis on whole blood hemolysates at pH 6.3 provides good resolution of HbA and HbA₁. The gel contains negatively charged moieties that interacts with the hemoglobin. After 25 to 35 minutes, the GHb separates on the cathodic side of HbA. Quantification is done by scanning densitometry at 415 nm.

The hemoglobin variant separate on IEF on the basis of their migration in gel containing pH gradient on acrylamide gel slabs.

Immunoassay with the principle of immunoinhibition are used like ELISA where antibodies are raised and used to inhibit other fraction in one hand and capture and detection antibodies are used to determine HbA1c.

Affinity gel columns are used to separate GHb, which binds to the column, from the nonglycated fraction. M-Aminophenylboronic acid is immobilized by cross linking to beaded agarose or another matrix (e.g., glass fiber). The boronic acid reacts the cis-diol groups of glucose bound to Hb to form a reversible five member ring complex thus selectively holding the GHb on the column. The nonglycated Hb does not bind. Sorbitol is then added to elute the GHb. Absorbance of the bound and nonbound fractions measured at 415 nm is used to calculate the percentage of GHb. Nonglycated Hb does not bind and is removed In a wash step. The sorbitol competes for boronate binding sites. 

(Source: Tietz Clinical Chemistry, 4th Edition)

For borate affinity assay, packed blood cells are mixed with hemolysate reagent that contain borate buffer. Glycated Hb is assayed from this hemolysate.