The C-peptide suppression test by insulin-induced hypoglycaemia may give results that do not clearly distinguish all patients from controls, and its interpretation may be difficult, since the results depend on several parameters including gender and body mass index (Service et al. 1992). A more sophisticated C-peptide suppression test, combined with the euglycaemic clamp, was successfully employed for the diagnosis of insulinoma, but it

\* Regarding the diagnostic criteria for inappropriate insulin secretion, previously employed ratios (insulin/glucose ratio, Turner's index) are no more in use and do not bring greater diagnostic accuracy than the presently-used criteria (Vezzosi et al. 2007) (Service 1995). Though insulinoma secrete a high proportion of proinsulin, the proinsulin/insulin ratio

considered as a tool to provide diagnostic evidence for a hypoglycaemic disorder.

concentrations below 3.3 mmol/L (0.6 g/L) during the fast test (O'Brien et al. 1993).

insulinoma in patients with a negative fast test (Wiesli et al. 2004b; Soh & Kek 2010). **Other diagnostic criteria for hypoglycaemia related to endogenous hyperinsulinism** 

2009).

could be considered.

use and has been replaced by the 72 hour-fast test.

cannot be recommended for clinical practice (Gin et al. 1987).

100% (O'Brien et al. 1993). There is no study specifying the validity of this criterion for another range of plasma glucose concentrations. Placzowski et al. reported 100% sensitivity and specificity for the diagnosis of insulinoma with beta-hydroxy-butyrate levels of 2.7 mmol/L or less during a fast test with concomitant plasma glucose concentrations below 0.6 g/L or below 0.5 g/L (Placzkowski et al. 2009), but the results are not detailed. There are two case reports of insulinoma patients with false negative results of beta-hydroxy-butyrate levels: an 80-year-old female patient with plasma glucose concentrations of 0.48 g/L (2.7 mmol/L), serum C-peptide of 0.93 ng/mL (0.31 nmol/L) and beta-hydroxy-butyrate levels of 3.0 mmol/L after 72 hours of fast (Wiesli et al. 2004b) and a 49-year-old man with plasma glucose concentrations of 0.32 g/L, serum C-peptide levels of 1.2 ng/mL, and betahydroxy-butyrate levels of 3.2 mmol/L (Soh & Kek 2010). Among our patients (Vezzosi et al. 2007), one patient with a malignant insulinoma was found to have, during a fast test, plasma glucose concentrations of 0.36 g/L, serum C-peptide levels of 1.7 ng/mL, plasma proinsulin levels of 37 pmol/L (which led to the unequivocal diagnosis of inappropriate insulin secretion) and plasma beta-hydroxy-butyrate levels of 3.8 mmol/L. We hypothesized that severe hypoglycaemia below 0.45 g/L could result in a greater secretion of counterregulatory hormones thereby leading to an increase in beta-hydroxy-butyrate levels; lipolysis may escape the inhibitory effect of insulin during hyperinsulinic hypoglycaemia (Lucidi et al. 2010). However we observed a patient with a malignant insulinoma whose lowest plasma glucose concentration during a 72 hour-fast test was 0.53 g/L after 58 hours of fast, with concomitant serum levels of insulin, C-peptide and proinsulin of 13 mIU/L, 2.4 ng/mL, and 36 pmol/L (which gave diagnostic evidence), respectively, and plasma beta-hydroxy-butyrate levels above 2.7 mmol/L. At the end of the 72 hour-fast test, beta-hydoxy-butyrate levels reached the value of 7.0 mmol/L in this patient, with concomitant concentrations of 0.68 g/L for plasma glucose, 4.0 mIU/L for serum insulin, 1.2 ng/mL for serum C-peptide and 25 pmol/L for serum proinsulin (personal data).

In conclusion, though being very valuable, the threshold of 2.7 mmol/L or less for betahydroxy-butyrate levels does not yield 100% sensitivity and specificity. Based on the observed results there are several caveats: 1) beta-hydroxy-butyrate levels may lose part of their diagnostic accuracy when plasma glucose concentrations are below 0.45 g/L and are of minor interest for this range of glucose levels; 2) proinsulin levels must be taken into account before ruling out the diagnosis of insulinoma in patients whose plasma glucose concentrations are within the 0.45-0.60 g/L range even when plasma beta-hydroxy-butyrate levels reach values above 2.7 mmol/L; 3) patients with recurrent insulinoma or nesidioblastosis after partial pancreatectomy may not fulfil the usual diagnostic criteria for insulin-mediated hypoglycaemia during the fast test. Whether a disruption in the progressive elevation of beta-hydroxy-butyrate levels concomitant with lower glucose levels during the fast test (with an insulin secretory burst) should lead to suspicion of insulinoma remains to be determined.

#### **Glucagon test**

According to the 2009 expert consensus (Cryer et al. 2009) an increase in plasma glucose of at least 25 mg/dL (0.25 g/L, 1.4 mmol/L) after intravenous glucagon indicate mediation of hypoglycaemia by insulin. This refers to the evaluation protocol established by the Mayo Clinic group (O'Brien et al. 1993) : the glucagon test is performed at the end of a prolonged fast test, i.e. either at the time of occurrence of symptomatic hypoglycaemia with concomitant

100% (O'Brien et al. 1993). There is no study specifying the validity of this criterion for another range of plasma glucose concentrations. Placzowski et al. reported 100% sensitivity and specificity for the diagnosis of insulinoma with beta-hydroxy-butyrate levels of 2.7 mmol/L or less during a fast test with concomitant plasma glucose concentrations below 0.6 g/L or below 0.5 g/L (Placzkowski et al. 2009), but the results are not detailed. There are two case reports of insulinoma patients with false negative results of beta-hydroxy-butyrate levels: an 80-year-old female patient with plasma glucose concentrations of 0.48 g/L (2.7 mmol/L), serum C-peptide of 0.93 ng/mL (0.31 nmol/L) and beta-hydroxy-butyrate levels of 3.0 mmol/L after 72 hours of fast (Wiesli et al. 2004b) and a 49-year-old man with plasma glucose concentrations of 0.32 g/L, serum C-peptide levels of 1.2 ng/mL, and betahydroxy-butyrate levels of 3.2 mmol/L (Soh & Kek 2010). Among our patients (Vezzosi et al. 2007), one patient with a malignant insulinoma was found to have, during a fast test, plasma glucose concentrations of 0.36 g/L, serum C-peptide levels of 1.7 ng/mL, plasma proinsulin levels of 37 pmol/L (which led to the unequivocal diagnosis of inappropriate insulin secretion) and plasma beta-hydroxy-butyrate levels of 3.8 mmol/L. We hypothesized that severe hypoglycaemia below 0.45 g/L could result in a greater secretion of counterregulatory hormones thereby leading to an increase in beta-hydroxy-butyrate levels; lipolysis may escape the inhibitory effect of insulin during hyperinsulinic hypoglycaemia (Lucidi et al. 2010). However we observed a patient with a malignant insulinoma whose lowest plasma glucose concentration during a 72 hour-fast test was 0.53 g/L after 58 hours of fast, with concomitant serum levels of insulin, C-peptide and proinsulin of 13 mIU/L, 2.4 ng/mL, and 36 pmol/L (which gave diagnostic evidence), respectively, and plasma beta-hydroxy-butyrate levels above 2.7 mmol/L. At the end of the 72 hour-fast test, beta-hydoxy-butyrate levels reached the value of 7.0 mmol/L in this patient, with concomitant concentrations of 0.68 g/L for plasma glucose, 4.0 mIU/L for serum insulin, 1.2 ng/mL for serum C-peptide and 25

In conclusion, though being very valuable, the threshold of 2.7 mmol/L or less for betahydroxy-butyrate levels does not yield 100% sensitivity and specificity. Based on the observed results there are several caveats: 1) beta-hydroxy-butyrate levels may lose part of their diagnostic accuracy when plasma glucose concentrations are below 0.45 g/L and are of minor interest for this range of glucose levels; 2) proinsulin levels must be taken into account before ruling out the diagnosis of insulinoma in patients whose plasma glucose concentrations are within the 0.45-0.60 g/L range even when plasma beta-hydroxy-butyrate levels reach values above 2.7 mmol/L; 3) patients with recurrent insulinoma or nesidioblastosis after partial pancreatectomy may not fulfil the usual diagnostic criteria for insulin-mediated hypoglycaemia during the fast test. Whether a disruption in the progressive elevation of beta-hydroxy-butyrate levels concomitant with lower glucose levels during the fast test (with an insulin secretory burst) should lead to suspicion of insulinoma

According to the 2009 expert consensus (Cryer et al. 2009) an increase in plasma glucose of at least 25 mg/dL (0.25 g/L, 1.4 mmol/L) after intravenous glucagon indicate mediation of hypoglycaemia by insulin. This refers to the evaluation protocol established by the Mayo Clinic group (O'Brien et al. 1993) : the glucagon test is performed at the end of a prolonged fast test, i.e. either at the time of occurrence of symptomatic hypoglycaemia with concomitant

pmol/L for serum proinsulin (personal data).

remains to be determined.

**Glucagon test** 

plasma glucose concentrations of 0.45 g/L (2.5 mmol/L) or less, or after 72 hours of fast: plasma glucose responses to the intravenous injection of 1 mg glucagon are measured at 10 min-intervals for 30 min. Under these conditions, plasma glucose response to glucagon was found to be higher in insulinoma patients than in controls with a range of maximal increase in plasma glucose concentrations of 1.4-5.4 mmol/L in insulinoma patients and of 0.1-1.3 mmol/L in controls, as a consequence of a lesser depletion of hepatic glycogen in insulinoma patients during the fast, which is related to the higher insulin secretion in the patients. This allowed a clear distinction between patients and controls who has reached plasma glucose concentrations below 3.3 mmol/L (0.6 g/L) during the fast test (O'Brien et al. 1993).

On the other hand, the glucagon test has long been known to be a provocative test in insulinoma patients: glucagon may trigger severe hypoglycaemia related to insulin secretion in insulinoma patients. This provocative test was found to be helpful for the diagnosis of insulinoma in patients with a negative fast test (Wiesli et al. 2004b; Soh & Kek 2010).

#### **Other diagnostic criteria for hypoglycaemia related to endogenous hyperinsulinism**

\* Regarding the various tests employed in the evaluation of a patient with suspected hypoglycaemic disorder, only the fast test, with a glucagon test at its termination, in patients with symptoms of fasting hypoglycaemia, and the mixed meal test in patients with postprandial hypoglycaemia are recommended by the 2009 expert consensus (Cryer et al. 2009).
