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Monitoring of Patients with Diabetes




Guidelines & Recommendations for Laboratory Analysis in the Diagnosis & Management of Diabetes Mellitus

Ketone Testing

David Goldstein, MD
University of Missouri
Columbia, MO

I.                     Use

Recommendation: Ketones should be measured in urine by both patients with diabetes and in the clinic/hospital setting as an adjunct for the diagnosis of diabetic ketoacidosis.  Only specific determinations of beta-hydroxybutyrate in blood should be used to establish a definitive diagnosis of diabetic ketoacidosis and to facilitate ongoing monitoring of ketoacidosis.

The ketone bodies, acetoacetate (ACAC), acetone (AN), and beta-hydroxybutyrate (BHBA), are catabolic products of triglycerides.  Determinations of ketones in urine and blood are widely used in the management of patients with diabetes mellitus as adjuncts for both diagnosis and ongoing monitoring of diabetic ketoacidosis (DKA).  Measurement of ketone bodies is routinely performed both in an office/hospital setting and at home by patients.

The American Diabetes Association (ADA) recommends that initial evaluation of patients with diabetes mellitus include determination of urine ketones and that urine ketone testing should be available in the physician’s office for immediate use as needed (1).  The ADA further recommends that urine ketone testing is an important part of monitoring by patients with diabetes, particularly in those with type 1 diabetes, pregnancy with preexisting diabetes, and gestational diabetes.  The ADA recommends that all people with diabetes (including those with type 2 diabetes) test their urine for ketones during acute illness or stress or when blood glucose levels are consistently elevated (e.g., >300 mg/dL[>16.7 mmol/L]), during pregnancy, or when any symptoms of ketoacidosis, such as nausea, vomiting, or abdominal pain, are present (2).  The ADA recommends that only specific determinations of BHBA in blood be used for diagnosis and ongoing monitoring of diabetic ketoacidosis rather than urine ketone testing or blood ketone testing that does not quantify BHBA (2).

II.                   Rationale

Ketone bodies are normally present in urine and blood but in very low concentrations.  Elevated levels in patients with known diabetes mellitus or in previously undiagnosed patients presenting with hyperglycemia suggest impending or established DKA, a medical emergency.  The two major mechanisms for the elevated ketone levels in diabetes mellitus are increased production from triglycerides and decreased utilization in the liver, both a result of absolute or relative insulin deficiency and elevated counterregulatory hormones including cortisol, epinephrine, glucagon, and growth hormone (3,4).

The principal ketone bodies BHBA and ACAC are normally present in approximately equimolar amounts.  AN is normally present in only small quantities, and is derived from spontaneous decarboxylation of ACAC.  The proportion of BHBA is appreciably increased in any condition that alters the redox state of hepatic mitochondria such as hypoxia, fasting, metabolic disorders, including DKA, and alcoholic ketoacidosis (5-7).  Thus, assay methods for ketones that do not include measurement of BHBA provide, at best, only limited, and at times, misleading clinical information (2,8).

III.         Analytical

A.     Urine ketones

1.      Pre-analytical: Normally, the concentrations of ketones in the urine are below the limits of detectability with commercially available testing materials.  False-positive results have been reported with highly colored urine and in the presence of several sulfhydryl drugs (9).  The urine test reagents deteriorate with exposure to air, giving false-negative readings; testing material should be stored in tightly sealed containers and discarded after the expiration date on the manufacturer label (10).  False-negative readings are also reported with highly acidic urine specimens, such as after large intakes of ascorbic acid. Loss of ketones in urine attributable to microbial action can also cause false-negative readings.  Since AN is a highly volatile substance, specimens should be kept in a closed container to prevent loss from evaporation.  For point-of-care analyses in medical facilities and for patients in the home setting, control materials (giving both negative and positive readings) are not commercially available but would be desirable to assure accuracy of test results.

2.      Analytical: Several assay principles have been described.  Most commonly used is the colorimetric reaction that occurs between ketones and nitroprusside (sodium nitroferricyanide) resulting in a purple color.  This method is widely available in the form of dipsticks and tablets and is used to measure ketones in both urine and blood (either serum or plasma).  Several manufacturers offer dipsticks that measure glucose and ketones; a combination dipstick is necessary only if the patient monitors urine glucose instead of or in addition to blood glucose.  The nitroprusside method measures only ACAC unless the reagent contains glycine, in which case AN is also measured.  The nitroprusside-containing reagent is much more sensitive to ACAC than AN with respect to color generation.  This method does not measure BHBA(2).

B.     Blood ketones

1.      Preanalytical:  Serum/plasma ketones can be measured using tablets or dipsticks routinely used for urine ketone determinations.  Although specimens can be diluted with saline to “titer” the ketone level (results are typically reported as “positive at a 1/x dilution”), as with urine ketone testing, BHBA, the predominant ketone body in diabetic ketoacidosis, is not detected.  For specific determinations of BHBA, as described below, specimen requirements differ among methods.  In general, blood samples can be collected into heparin, EDTA, fluoride, citrate or oxalate (for the BioScanner Ketone system, fluoride and oxalate have not been tested according to the manufacturer).  Ascorbic acid interferes with some assay methods.  ACAC interferes with some assay methods unless specimens are highly dilute.  Specimen stability differs among methods but in general, whole blood specimens are stable at 4 degrees C for up to 24 hours, and serum/plasma specimens for up to one week at 4 degrees C and for at least several weeks at –20 degrees C (long-term stability data are not available for most assay methods).

2.   Analytical

Although a number of different types of assay methods for blood ketones, including specific measurement of BHBA have been described (e.g., colorimetric, gas chromatography, capillary electrophoresis, enzymatic), enzymatic methods for quantification of BHBA are most widely used for routine clinical management (11-13).  The principle of the enzymatic methods is that BHBA in the presence of NAD is converted to ACAC and NADH by B-hydroxybutyrate dehydrogenase.  Under alkaline conditions (pH 8.5-9.5), the reaction favors formation of ACAC from BHBA.  The NADH produced can be quantified spectrophotometrically (Sigma, St. Louis, MO) or kinetically using a peroxidase reagent (Analox Instruments U.S.A., Lunenburg, MA)  One manufacturer offers a method that utilizes a test card impregnated with the reagents (KetoSite, GDS Diagnostics, Elkhart, IN).  Most methods permit use of whole blood, plasma, or serum specimens (required volumes are generally 200 microliters or less).  Some methods permit analyses of multiple analytes and are designed for point-of-care testing.  Several methods are available as hand-held meters, which are FDA-approved for both laboratory use and for over-the-counter use by patients (e.g., BioScanner Ketone, PolymerTechnology Systems, Indianapolis, IN; MediSense Precision Xtra, Abbott Laboratories, Abbott Park, IL)(13).  These methods utilize dry chemisty test strips to which a drop of whole blood, serum, or plasma is added.  Results are displayed on the instruments within approximately 2 minutes.

III.                  Interpretation

A.     Urine ketone determinations

Recommendations:  Urine ketones should be measured by all patients with diabetes mellitus according to the ADA recommendations.  Urine ketones should be measured by health-care providers only as an adjunct to diagnosis of diabetic ketoacidois and should not be used to monitor therapy of diabetic ketoacidosis

The ADA recommends that all patients with diabetes mellitus test their urine for ketones during acute illness or stress or with persistent hyperglycemia (plasma glucose >300 mg/dlL or 16.7 mmol/L), during pregnancy, or when any symptoms of DKA, such as nausea, vomiting, or abdominal pain, are present(1,2,8).  In a patient with known diabetes mellitus or in a patient not previously diagnosed with diabetes but who presents with typical symptoms of diabetes and hyperglycemia, the presence of positive urine ketone readings suggests the possibility of impending or established DKA.  Although DKA is most commonly associated with type 1 diabetes mellitus, it may occur in type 2 patients(14).  Patients with alcoholic ketoacidosis will have positive urine ketone readings but hyperglycemia is not usually present.  Positive urine ketone readings are found in up to 30% of first morning urine specimens from pregnant women (diabetic or not), during starvation, and after hypoglycemia(2,8,15).  Urine ketone determinations should not be used to monitor the course of DKA since they do not measure the predominant ketone BHBA and may show increased levels suggesting  failure of therapy even though BHBA levels in the blood are decreasing from oxidation to ACAC(2-8).

B.     Blood ketone determinations

Recommendations:  Blood ketone determinations that rely on the nitroprusside reaction should be used only as an adjunct to diagnosis of diabetic ketoacidosis and should not be used to monitor treatment of diabetic ketoacidosis.  Specific measurement of BHBA in blood can be used for diagnosis and monitoring of diabetic ketoacidosis but further studies are needed to determinine if the test offers any advantage over more traditional approaches (e.g., measurements of serum CO2, pH).  Further studies are also needed to determine if blood ketone determinations by patients with diabetes mellitus are preferable (e.g., better accepted by patients than urine testing, more prompt diagnosis of diabetic ketoacidosis) to urine ketone determinations as currently recommended by the ADA.

Blood ketone determinations that rely on the nitroprusside reaction should be used with caution for diagnosis of DKA as results do not quantify BHBA the predominant ketone in DKA).  The test should not be used to monitor the course of therapy since levels of ACAC and AN may rise as levels of BHBA fall during successful therapy(2-8).  Blood ketone determinations that measure BHBA specifically are useful for both diagnosis and ongoing monitoring of DKA(7,16-18). However, only a limited number of studies have been performed comparing measurements of BHBA, CO2/HCO3, pH, etc. in patients with DKA.  It is not known if BHBA determinations offer any substantive advantage over more traditional (e.g., clinical judgment, serum/plasma CO2, pH) approaches to diagnosis and monitoring patients with DKA.  Reference intervals for BHBA differ among assay methods but levels in healthy individuals fasted overnight are generally <0.5 mmol/L  Levels in patients with well-documented diabetic ketoacidosis (serum CO2 <17 mmol/L, arterial pH <7.3, plasma glucose >250 mg/dL are generally >2 mm/L.


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