a. Glucosuria - A specific and convenient method to detect glucosuria is the paper strip impregnated with glucose oxidase and a chromogen system (Clinistix, Diastix), which is sensitive to as little as 0.1% glucose in urine. Diastix can be directly applied to the urinary stream, and differing color responses of the indicator strip reflect glucose concentration.
A normal renal threshold for glucose as well as reliable bladder emptying is essential for interpretation.
b. Ketonuria - Qualitative detection of ketone bodies can be accomplished by nitroprusside tests (Acetest or Ketostix). Although these tests do not detect β-hydroxybutyric acid, which lacks a ketone group, the semiquantitative estimation of ketonuria thus obtained is nonetheless usually adequate for clinical purposes.
Blood testing procedures
a. Glucose tolerance test -
(1) Methodology and normal fasting glucose - Plasma or serum from venous blood samples has the advantage over whole blood of providing values for glucose that are independent of hematocrit and that reflect the glucose concentration to which body tissues are exposed. For these reasons, and because plasma and serum are more readily measured on automated equipment, they are used in most laboratories. If serum is used or if plasma is collected from tubes that lack an agent to block glucose metabolism (such as fluoride), samples should be refrigerated and separated within 1 hour after collection. The glucose concentration is 10-15% higher in plasma or serum than in whole blood because structural components of blood cells are absent.
(2) Criteria for laboratory confirmation of diabetes mellitus - If the fasting plasma glucose level is 126 mg/dL or higher on more than one occasion, further evaluation of the patient with a glucose challenge is unnecessary. However, when fasting plasma glucose is less than 126 mg/dL in suspected cases, a standardized oral glucose tolerance test may be done.
For proper evaluation of the test, the subjects should be normally active and free from acute illness. Medications that may impair glucose tolerance include diuretics, contraceptive drugs, glucocorticoids, niacin, and phenytoin.
Because of difficulties in interpreting oral glucose tolerance tests and the lack of standards related to aging, these tests are being replaced by documentation of fasting hyperglycemia.
b. Glycated hemoglobin (hemoglobin A1) measurements - Hemoglobin becomes glycated by ketoamine reactions between glucose and other sugars and the free amino groups on the α and β chains. Only glycation of the N-terminal valine of the beta chain imparts sufficient negative charge to the hemoglobin molecule to allow separation by charge dependent techniques. These charge separated hemoglobins are collectively referred to as hemoglobin A1 (HbA1). The major form of HbA1 is hemoglobin A1c (HbA1c) where glucose is the carbohydrate. HbA1c comprises 4-6% of total hemoglobin A1. The remaining HbA1 species contain fructose-1,6 diphosphate (HbA1a1); glucose-6-phosphate (HbA1a2); and unknown carbohydrate moiety (HbA1b). The hemoglobin A1c fraction is abnormally elevated in diabetic persons with chronic hyperglycemia. Methods for measuring HbA1c include electrophoresis, cation-exchange chromatography, boronate affinity chromatography, and immunoassays. Office-based immunoassays using capillary blood give a result in about 9 minutes and this allows for immediate feedback to the patients regarding their glycemic control.
Since glycohemoglobins circulate within red blood cells whose life span lasts up to 120 days, they generally reflect the state of glycemia over the preceding 8-12 weeks, thereby providing an improved method of assessing diabetic control. The HbA1c value, however, is weighted to more recent glucose levels (previous month) and this explains why significant changes in HbA1c are observed with short-term (1 month) changes in mean plasma glucose levels. Measurements should be made in patients with either type of diabetes mellitus at 3- to 4-month intervals so that adjustments in therapy can be made if HbA1c is either subnormal or if it is more than 2% above the upper limits of normal for a particular laboratory. In patients monitoring their own blood glucose levels, HbA1c values provide a valuable check on the accuracy of monitoring. In patients who do not monitor their own blood glucose levels, HbA1c values are essential for adjusting therapy. Data from the Diabetes Control and Complications Trial (DCCT) showed that there is a linear relationship between the HbA1c and the mean of seven-point capillary blood glucose profiles (preprandial, postprandial, and bedtime). Thus, mean plasma glucose levels of 170, 205, 240, and 275 mg/dL approximately correlate with HbA1c values of 7%, 8%, 9%, and 10%, respectively. Use of HbA1c for screening is controversial. Sensitivity in detecting known diabetes cases by HbA1c measurements is only 85%, indicating that diabetes cannot be excluded by a normal value. On the other hand, elevated HbA1c assays are fairly specific (91%) in identifying the presence of diabetes.
The accuracy of HbA1c values can be affected by hemoglobin variants or derivatives; the effect depends on the specific hemoglobin variant or derivative and the specific assay used. Immunoassays that use an antibody to the glycated amino terminus of β globin do not recognize the terminus of the γ globin of hemoglobin F. Thus, in patients with high levels of hemoglobin F, immunoassays give falsely low values of HbA1c. Cation-exchange chromatography separates hemoglobin species by charge differences. Hemoglobin variants that co-elute with HbA1c can lead to an overestimation of the HbA1c value. Chemically modified derivatives of hemoglobin such as carbamoylation (in renal failure) or acetylation (high-dose aspirin therapy) can similarly co-elute with HbA1c by some assay methods.
Any condition that shortens erythrocyte survival or decreases mean erythrocyte age (eg, recovery from acute blood loss, hemolytic anemia) will falsely lower HbA1c irrespective of the assay method used. Alternative methods such as fructosamine should be considered for these patients. Vitamins C and E are reported to falsely lower test results possibly by inhibiting glycation of hemoglobin.
c. Serum fructosamine - Serum fructosamine is formed by nonenzymatic glycosylation of serum proteins (predominantly albumin). Since serum albumin has a much shorter half-life than hemoglobin, serum fructosamine generally reflects the state of glycemic control for only the preceding 1-2 weeks. Reductions in serum albumin (eg, nephrotic state or hepatic disease) will lower the serum fructosamine value. When abnormal hemoglobins or hemolytic states affect the interpretation of glycohemoglobin or when a narrower time frame is required, such as for ascertaining glycemic control at the time of conception in a diabetic woman who has recently become pregnant, serum fructosamine assays offer some advantage. Normal values vary in relation to the serum albumin concentration and are 1.5-2.4 mmol/L when the serum albumin level is 5 g/dL.
d. Self-monitoring of blood glucose - Capillary blood glucose measurements performed by patients themselves, as outpatients, are extremely useful. In type 1 patients in whom “tight” metabolic control is attempted, they are indispensable. There are several paper strip (glucose oxidase, glucose dehydrogenase, or hexokinase) methods for measuring glucose on capillary blood samples. A reflectance photometer or an amperometric system is then used to measure the reaction that takes place on the reagent strip. A large number of blood glucose meters are now available. All are accurate, but they vary with regard to speed, convenience, size of blood samples required, and cost. Popular models include those manufactured by LifeScan (One Touch), Bayer Corporation (Glucometer Elite, DEX), Roche Diagnostics (Accu-Chek), Abbott Laboratories (ExacTech, Precision, FreeStyle), and Home Diagnostics (Prestige). A Freestyle Flash meter, for example, requires only 0.3 mL of blood and gives a result in 7 seconds - and illustrates how there has been continued progress in this technologic area. Various glucometers appeal to a particular consumer need and are relatively inexpensive, ranging from $50.00 to $100.00 each. The more expensive models compute blood glucose averages and can be attached to printers for data records and graph production. Test strips remain a major expense, costing 50-75 cents apiece. In self-monitoring of blood glucose, patients must prick a finger with a 28 to 30 gauge lancets, which can be facilitated by a small plastic trigger device such as an Autolet (Ames Co.), SoftClix (Boehringer-Mannheim), or Penlet (Lifescan, Inc.). When used for multiple patients, as in a clinic, physician’s office, or hospital ward, disposable finger-rest platforms are required to avoid inadvertent transmission of blood-borne viral diseases. Some meters such as the FreeStyle (Abbott Laboratories) have been approved for measuring glucose in blood samples obtained at alternative sites such as the forearm and thigh. There is, however, a 5- to 20-minute lag in the glucose response on the arm with respect to the glucose response on the finger. Forearm blood glucose measurements could therefore result in a delay in detection of rapidly developing hypoglycemia.
The clinician should be aware of the limitations of the self-monitoring glucose systems. First, a few of the older meters (such as the One Touch Profile) are calibrated against whole blood glucose concentrations even though the test strip measures the glucose in the plasma fraction. This means the displayed values are 10% to 15% lower than the laboratory glucose result. Second, increases or decreases in hematocrit can decrease or increase the measured glucose values. The mechanism underlying this effect is not known but presumably it is due to the impact of red cells on the diffusion of plasma into the reagent layer. Third, the meters and the test strips are calibrated over the glucose concentrations ranging from 60 mg/dL to 160 mg/dL, and the accuracy is not as good for higher and lower glucose levels. When the glucose is less than 60 mg/dL, the difference between the meter and the laboratory value may be as much as 20%. Fourth, glucose oxidase-based amperometric systems underestimate glucose levels in the presence of high oxygen tension. This may be important in the critically ill who are receiving supplemental oxygen; under these circumstances, a glucose dehydrogenase-based system may be preferable. The accuracy of data obtained by glucose monitoring requires education of the patient in sampling and measuring procedures as well as in proper calibration of the instruments. Bedside glucose monitoring in a hospital setting requires rigorous quality control programs and certification of personnel to avoid errors.
e. Continuous glucose monitoring systems - Two continuous glucose monitoring systems are currently available for clinical use. The system manufactured by Medtronic Minimed involves inserting a subcutaneous sensor (rather like an insulin pump cannula) that measures glucose concentrations in the interstitial fluid for 72 hours. In the newest version of the system (Guardian RT), the glucose values are available for review by the patient at the time of measurement. There are also options to set alarms for dangerously low or high glucose values. The other system (“Glucowatch”) measures glucose in interstitial fluid extracted through intact skin by applying a low electric current (reverse iontophoresis). This process can cause local skin irritation, and sweating distorts the glucose measurement. Both systems require calibration with finger blood glucose measurements. The main value of these systems appears to be in identifying episodes of asymptomatic hypoglycemia, especially at night.
Lipoprotein abnormalities in diabetes
- Circulating lipoproteins are just as dependent on insulin as is the plasma glucose. In type 1 diabetes, moderately deficient control of hyperglycemia is associated with only a slight elevation of LDL cholesterol and serum triglycerides and little if any change in HDL cholesterol. Once the hyperglycemia is corrected, lipoprotein levels are generally normal. However, in obese patients with type 2 diabetes, a distinct “diabetic dyslipidemia” is characteristic of the insulin resistance syndrome. Its features are a high serum triglyceride level (300-400 mg/dL), a low HDL cholesterol (less than 30 mg/dL), and a qualitative change in LDL particles, producing a smaller dense particle whose membrane carries supranormal amounts of free cholesterol. These smaller dense LDL particles are more susceptible to oxidation, which renders them more atherogenic. Since a low HDL cholesterol is a major feature predisposing to macrovascular disease, the term “dyslipidemia” has preempted the term “hyperlipidemia,” which mainly denoted the elevated triglycerides. Measures designed to correct the obesity and hyperglycemia, such as exercise, diet, and hypoglycemic therapy, are the treatment of choice for diabetic dyslipidemia, and in occasional patients in whom normal weight was achieved, all features of the lipoprotein abnormalities cleared. Since primary disorders of lipid metabolism may coexist with diabetes, persistence of lipid abnormalities after restoration of normal weight and blood glucose should prompt a diagnostic workup and possible pharmacotherapy of the lipid disorder. Chapter 28 discusses these matters in detail.