Extracción de características

The biochemical and physiological changes in glucose metabolism that occur during a fast have already been outlined in chapter 1. The diagnostic fast was the standard

provocation test used to induce hypoglycaemia (Morris AAM et al 1996). The aim of the fast was to induce a length of period without food so that the physiological mechanisms regulating glycogenolysis, gluconeogensis, fetty acid oxidation and ketone body

formation are normally switched on. If there was a defect in any o f these pathways this should lead to hypoglycaemia during the fast.

This test was carefully explained to all the parents/children who participated in the study. Prior to the start of the diagnostic fast each child had an intraveous cannula inserted or in the case of the neonates a central venous line such as a Hickman line under general anaesthesia in the operating theatre was inserted. The child was then fasted according to unit protocol. The length of this was determined by the age o f the child (Table 3.4). The fast involved stopping all enteral and intravenous fluids. In the older children blood glucose (BM stix and laboratory blood glucose) was measured hourly to begin with and at half hourly intervals if hypoglycaemia was anticipated. In the neonates blood glucose was measured at 10-15 minute intervals. The fast was terminated if the laboratory blood glucose concentration was <2.6mmol/L or if the child showed symptoms of

hypoglycaemia (drowsy, confusion, nausea, vomiting, headaches). During the fast each of the infants and children were closely monitored by investigator for signs and

symptoms of hypoglycaemia. In the older children blood was withdrawn for serum cortisol and GH measurements at the beginning of the fast, at hourly intervals throughout

the fast, at the time of hypoglycaemia and at 10 minute intervals for 50 minutes after the hypoglycaemic stimulus was corrected. The 10 minute sampling interval was chosen in order to detect pulses of GH secretion during a total sampling interval of 50 minutes from the time of the hypoglycaemia. The hypoglycaemia was corrected either with

intravenous fluids (1 - 2 ml of 1 0% dextrose bolus followed by a continuous infusion of

dextrose to maintain normoglycaemia) or oral feeds if the child was tolerating enteral feeding.

In the neonatal group blood was drawn from the central line for serum cortisol and GH measurements at the beginning of the fast, at 1 0 minute intervals during the fast and then

at the time of hypoglycaemia. The hypoglycaemia was then corrected with l-2ml/kg of

1 0% dextrose followed by a continuous infusion of dextrose to maintain

normoglycaemia. Blood for serum cortisol and GH measurements was again withdrawn at ten minute intervals for fifty minutes after correction of the hypoglycaemia.

Six of the neonates with HI had serum ACTH measured at the time of hypoglycaemia. They were then given 62.5micrograms of Synacthen intravenously after correction of the hypoglycaemia. Blood was withdrawn again at ten minute intervals for fifty minutes after correction of the hypoglycaemia for serum cortisol measurement.

None of the children and neonates fasted were hypoglycaemic forty eight hours before the diagnostic fast was performed. Prior to the fast normoglycaemia was maintained in all the neonates using a combination of concentrated dextrose infusion and enteral feeds.

In total approproximately 3 mis of blood was taken for the complete hypoglycaemic profile at the time of hypoglycaemia. The intermediary metabolites measured at the beginning of the fast and at the time of hypoglycaemia are shown in table 3.5. At the bedside the blood samples for acetoacetate and pyruvate were precipitated by the addition of perchloric acid to the blood specimen. The ACTH sample was collected into cold EDTA tubes and immediately transported to the laboratory. All the other specimens were then transported to the Biochemistry Laboratory at Great Ormond Street Hospital. The GH and cortisol blood samples were centrifuged for three minutes, separated and serum stored at -20 until analysed. The blood samples for plasma glucose, lactate and pyruvate were centrifuged and plasma separated for analysis immediately. The blood samples for plasma NEFA, Ketone bodies, amino acids and ACTH were centrifuged, separated and serum stored at - 2 0 until analysis.

The m axim um length o f fast according to the age o f the child.

Age Length o f fast (hours)

0 - 6 months 6 6 - 8 months 8 8 - 1 2 months 1 2 1- 2 years 16 2 - 8 years 18 > 8 years 2 0

Table 3.5 Length of fast according to age of child. The length of the diagnostic fast was performed according to the age of the child. The neonatal group was fasted for less than six hours as they became hypoglycaemic very quickly upon withdrawal of exogenous glucose.

Metabolite Volume of blood sample (mis) Collected into (container)

Glucose 0.2 flouride

Insulin 0.5 lithium heparin

Cortisol 0.3 clotted

ACTH 0.5 ice cold EDTA

GH 0.5 clotted

Lactate 0.2 flouride

Pyruvate 0.2 precipitate by bedside

NEFA 0.1 lithium heparin

Acetoacetate 0.1 precipitate by bedside

P-hydroxy butyrate 0.1 lithium heparin

Branch chain amino acids 1ml lithium heparin

Urine organic acid 5 mis of urine

Table 3.6 The hormones and intermediary metabolites measured at the beginning of the fast and at the time of hypoglycaemia. Urine sample was collected at the end of the fast. Acyl-carnitine was measured on Guthrie card blood samples.

child was then fasted overnight prior to the test. On the morning o f the test at time -30 minutes a blood sample was taken for glucose, cortisol and GH. Insulin (0.1 units/kg) was then administrated intravenously. Blood samples for glucose, cortisol and growth hormone were again taken at +15, +30, +45, +60 and +90 minutes.