4.18: Diabetes, Ketone Analysis

A resident calls the laboratory, concerned about some apparently discordant laboratory results obtained for a 65 year old white female who came into the emergency room dehydrated and in moderate diabetic ketoacidosis. Laboratory results obtained following admission to the emergency room were as follows: pH, 7.17; PCO₂, 9 mmHg; glucose, 7832 mg/L; serum ketones (by dipstick), trace to small; potassium, 2.8 mmol/L; and sodium, 131 mol/L.

The patient was treated with fluids and insulin and she responded well, her glucose dropping to 1958 mg/L at 48 hours. What confuses the resident is that her serum ketone levels, after 48 hours of treatment, are now reported as moderate, although her glucose has decreased considerably. The resident questions whether the laboratory has mixed up or misanalyzed the specimen.

QUESTIONS

1. What can the laboratory do to verify the results?
2. The technologist retrieves the specimens in question and validates the patient’s identification number on the specimen tube. The technologist repeats the glucose and ketone body analysis, obtaining results very similar to the original ones. The technologist also notes that the earlier specimen was fairly turbid, while the later specimen was not. What explanation would you give to the resident for the increase in serum ketones following treatment?

Questions to Consider

1. What are “ketone bodies?”
2. Why are ketone bodies produced during diabetic ketoacidosis?
3. What are the pathways involved for the production of ketone bodies?
4. How are ketones measured?

Answer

1. If the laboratory has alternative methods for ketone analysis, the levels of acetoacetic acid and acetone can be verified. The results are verified if alternative tests are not available if the repeat analysis agrees with the initial results.
   
   First, check the patient identification on each phlebotomy tube for this patient to make sure that each was most likely drawn from the correct patient. Then, the dipstick analysis for each specimen is repeated to ensure that there was no analytical error.
2. The resident should not be concerned with the results. Early in severe DKA, the reactions described above are shifted strongly toward the production of NAD⁺ and \(\beta\)-hydroxybutyrate, with the measureable ketone, acetoacetic acid, assuming a realtively small proportion of the total 'ketone bodies'. (See Chapter 32 and Method Ketones on CD-ROM). Thus, the total amount of increased ketone bodies is greatly underestimated when measured by the nitroprusside reaction. Later, as the DKA is treated and resolved, the body begins to consume the \(\beta\)-hydroxybutyric acid and to metabolize it completely. In doing so, the reactions leading up to the production of beta-hydroxybuterate (Figure 32-9) are reversed, thus forming more acetoacetate in the process. Therefore, during the recovery phase (i.e. the second specimen in question) the apparent amount of ketones present increases, when in reality the total amount of ketones is decreasing; only a transient increase in acetoacetic acid is being detected. The turbidity of the specimen plays no significant part in the explanation of the ketone results.

Answers to Questions to Consider

1. “Ketone bodies” is a name given to a group of metabolites of fatty acid oxidation. They are acetoacetic acid, acetone, and \(\beta\)-hydroxybutyric acid. Only the first two are true ketones (See Chapter 32 and Method Ketones on CD-ROM).
2. During starvation or diabetic ketoacidosis (DKA) (Chapter 32), low insulin levels and relatively high hyerglycemic hormones (such as, cortisol) cause fat to be mobilized from adipose tissue, resulting in the release of free fatty acids. The fatty acids are oxidized by the liver to acetyl CoA and NADH. However, the large amounts of fatty acids being metabolized in ketoacidosis soon exceed the liver cells' capacity to recycle coenzyme A (CoA) and NAD⁺. This prevents additional metabolism of fatty acids. To allow continued fatty acid metabolism, the acetyl CoA reacts to form the ketone bodies and free CoA and NAD⁺ (Chapter 32).
3. The pathways of ketone body production are shown in Figure 32-9 page 594. Thus, the end-point of ketone body formation is \(\beta\)-hydroxybutyric acid and NAD+, which raises the NAD⁺/NADH ratio and allows continued fatty acid oxidation.
4. Routinely, serum or urine ketones are detected by dry reagent strip techniques which employ the nitroprusside reaction (See Method Ketones on CD-ROM). However, this reaction only detects acetoacetic acid and, to a much lesser extent, acetone. \(\beta\)-hydroxybutyric acid does not reactat all. Acetoacetic acid and \(\beta\)-hydroxybutyric acid can be quantitated by specific enzymatic reactions employing \(\beta\)-hydroxybutyrate dehydrogenase and measuring either the production or consumption of NADH (See Method Ketones on CD-ROM). The enzymatic assays are rarely available in a routine laboratory.