BACK TO INTRODUCTION
& GENETIC TESTING IN DIABETES
ROLE OF GLUCOSE MANAGEMENT IN DIABETES
& MINIMALLY INVASIVE GLUCOSE ANALYSIS
of Patients with Diabetes
& PRECURSORS, LEPTIN & AMYLIUN: IS THERE A ROLE?
FOR MICRO ALBUMINURIA
& Recommendations for Laboratory Analysis in the Diagnosis & Management
of Diabetes Mellitus
University of Missouri
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.
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).
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).
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
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.
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).
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).
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.
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
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).
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.
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
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