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β-Hydroxybutyrate Kit (Enzymatic Colorimetric Method)
Application
In vitro test for the quantitative determination of β-H Bconcentration in human serum or plasma on photometric systems.β-hydroxybutyrate (β-HB) is one of three sources of ketone bodies. Its relative proportion in the blood (78%) is greater than the other two ketone bodies, acetoacetate (20%) and acetone (2%). During carbohydrate deprivation (starvation, digestive disturbances, frequent vomiting), decreased carbohydrate utilization (diabetes mellitus), glycogen storage diseases, and alkalosis, acetoacetate production increases. The increase may exceed the metabolic capacity of the peripheral tissues. As acetoacetate accumulates in the blood, a small amount is converted to acetone by spontaneous decarboxylation. The remaining and greater portion of acetoacetate is converted to β-HB. In diabetics, β- HB measurements can be used for the assessment of the severity of diabetic coma and is essential for the exclusion of hyperosmolar non-ketotic diabetic coma. The measurement of β-HB is also used to monitor insulin requirements, based on existing hyperketonemia.
β -Hydroxybutyrate gets converted to acetoacetate and NADH in the presence of NAD by β-Hydroxybutyrate dehydrogenase. The NADH produced is converted to color using INT and diaphorase. The concentration is determined by measurement of the absorbance change at 505 nm due to INT.
Reagents
Sample Requirements
Calibrator Preparation
Carefully open the bottle, avoiding the loss of lyophilizate, and pipette in exactly 1.0 mL of distilled/deionized water. Carefully close the bottle and dissolve the contents completely by occasional gentle swirling within 30 minutes. Avoid the formation of foam. The dissolved calibrator can be used without any other Pretreatment.
Quality control Preparation
Carefully open the bottle, avoiding the loss of lyophilizate, and pipette in exactly 1.0 mL of distilled/deionized water. Carefully close the bottle and dissolve the contents completely by occasional gentle swirling within 30 minutes. Avoid the formation of foam. The dissolved control can be used without any other Pretreatment.
Method
Calibration
It is recommended to use the Calibrator from Hzymes and distilled/deionized water for two-point calibration. Calibration frequency: After reagent lot changed. As required following quality control procedures.
Quality control
At least two levels of control material should be analyzed with each batch of samples. Each laboratory should establish its own internal quality control scheme and procedures for ceptable tolerances.
Reference Intervals
Each laboratory should establish its own reference intervals based upon its patient population. The reference intervals measured at 37 °C listed below were taken from literature. Serum / Plasma: 0.03~0.30 mmol/L
Interferences/Specificity
The following substances were tested for interference with this methodology. Criterion: Recovery within ±10 % of initial value
Storage and stability
Up to expiration date indicated on the label, when stored unopened at 2-8℃ and protected from light. Once opened, the reagents are stable for 28 days when refrigerated on the analyzer or refrigerator. Contamination of the reagents must be avoided. Do not freeze the reagents. Once dissolved, the calibrator are stable for 7 days at 2– 8℃, the control are stable for 7 days at 2–8℃，do not freeze.
Reagent Blank Absorbance
The absorbance of β-HB reagent blank at 505 nm should be ＜0.4 A.
Sensitivity/Detection Limit
The lowest measurable β-HB concentration that can be distinguished from zero is 0.03 mmol/L with 99.7% confidence.
Precision
Within-run : CV≤5%
Between-run: CV≤10%
Linearity range
Conventional Units: 0.03~5.5 mmol/L If the value of sample exceeds 5.5 mmol/L, the sample should be diluted with 9 g/L NaCl solution (e.g. 1+3) and rerun; the result should be multiplied by 4. Assay urinary uric acid, the sample should be diluted 1 + 9 with 9 g/L NaCl and the result multiplied by 10.
Warnings and Precautions
1.For in vitro diagnostic use.
2.Take the necessary precautions for the use of laboratory reagents.
3.Preservative contained. Do not swallow. Avoid contact with skin and mucous membranes.
4.Disposal of all waste material should be in accordance with local guidelines.
5.Material safety data sheet is available on request for professional users.
【References】
[1] Ashwood ER. Tietz Textbook of Clinical Chemistry. Edited by CA Burtis. Philadelphia. WB Saunders Co. 1999.
[2] Vassault A, Bonnefont JP, Specola N, et al. Lactate, pyruvate, and ketone bodies. In Techniques in Diagnostic Human Biochemical Genetics – A Laboratory Manual. Edited by F Hommes. New York. Wiley-Liss. 1991.
[3] Bonnefont JP, Specola NB, Vassault A, et al. The fasting test in paediatrics: application to the diagnosis of pathological hypo- and hyperketotic states. Eur J Pediatr 1990;150:80-85.
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