Between July and November 2006, we sent invitation letters to 59 patients with (suspected) GS. After comprehensive information, 20 patients matched the inclusion criteria and gave their written informed consent. Two patients were excluded from the study, as they matched a stop criterion: One 28-year-old male with remittent ulcerative colitis had a haptoglobin level <0,2 due to a so far unknown slight haemolysis (haemoglobin concentration = 7.6 mmol/L, normal range 8.2–10.7; lactate dehydrogenase 3.82 microkat/l, normal range 2.25–3.75). A 50-year-old female was excluded due to a liver nodule of 1 centimetre. Invasive diagnostics and a six months follow-up did not reveale malignancy so far. Furthermore, one 32-year-old female declined to continue the study after the sonographic finding of a fatty liver.

The inverse starving test required an overnight fast. On arrival in the out-patients department, an initial blood sampling for conjugated and unconjugated bilirubin, ALAT, ASAT, GGT, lactate dehydrogenase, alkaline phosphatase and haptoglobine was done via an intravenous catheter. Within the next two hours, an abdominal ultrasound was carried out to exclude focal liver lesions and mechanic cholestasis. Two patients with abnormal findings in these investigations had to be excluded and were advised to undergo further diagnostics. 18 Patients without exclusion criteria got a free lunch of choice in the university restaurant after a second fasting blood sample was taken directly before lunch. After lunch, blood samples for bilirubin analysis were taken hourly up to 4 hours.

The nicotinic acid test was performed at the next day. After an overnight fast and fasting UCB analysis, the patients ingested 170 mg nicotinic acid with pure water. Further blood samples were taken in hourly intervals up to 4 hours. Between the repeated blood drawings via an intravenous catheter, the patients were advised to rest in the waiting room, and were only allowed to drink pure water.

18 GS patients completed both tests at two consecutive days. These included 11 males and 7 females with a median age of 34.5 years (range, 21–58), and a median body mass index of 22.1 kg/m² (range, 21.5–24). Two patients smoked 10 or 15 cigarettes per day, respectively, whereas the others were non-smokers. No patient regularly drinks alcohol, and none drunk alcohol within 24 hours before the tests and at the test days. None of the females used hormonal contraceptives. No patient had fever or felt unwell within one month before the tests.

The test patients had different initial symptoms. Yellow eyes (one with coincidental yellow skin) were noticed by three patients by themselves and in five cases by a relative. Fatigue was the first symptom in one case. In the remaining nine, elevated bilirubin levels were an incidental laboratory finding. The eight patients who consulted their doctors due to jaundice were significantly more likely to undergo liver biopsy (n = 4) or endoscopic retrograde cholangio-pancreaticography (ERCP, n = 1) than the nine patients with an incidental finding of elevated UCB (n = 0, p = 0.038, fisher exact test). Interestingly, fasting UCB was not different between these groups (p > 0.3, t-test).

Previously, all patients had ultrasound examinations; a computed tomography was done in three patients and magnet resonance tomography in two cases. Fasting UCB was not different between the patients who have undergone extensive radiological diagnostics and the patients with ultrasound only (p > 0.5). Bone marrow examination was not carried out in any case.

The inverse starving test

The median starving time between last caloric intake at the day before this test and the lunch of choice in the university restaurant was 15:30 hours (range: 13:30–17:15 hours). Side effects were recognized in two patients (table 3). As shown in figure 1, the inverse starving test induced an UCB reduction to normal range (<15.4 μmol/L) or a relative UCB reduction of more than 50% in none of the patients. Box plot analysis revealed only a negligible decrease in UCB levels over the test period (figure 2). We hypothesized that the UCB decline after lunch should be mutually dependent from caloric intake, but no correlation could be detected (Figures 2 and 3).

Adverse events of the inverse starving test
1 pt. headache at the evening of the test day
1 pt. vomiting at the afternoon and evening of the test day

The nicotinic acid test

Basal UCB at day 2 (nicotinic acid test) were almost identical (p = 0.88, t-test) with day 1 (inverse starving test). Two hours after ingestion of 170 milligrams NA, UCB was elevated > 50% in 15 of the 18 patients (sensitivity 83%, figure 4). Box plot analysis showed the typical course of nicotinic acid test with a precise discrimination of the relative UCB from the baseline value (figure 2). NA side effects were recognized in 16 patients (table 4). The conjugated bilirubin levels did not change during both tests (data not shown).

Adverse events of the nicotinic acid test
10 pts. itching skin sensations
9 pts. skin erythema
6 pts. heat waves
5 pts. burning skin sensations
3 pts. Vertigo
2 pts. Nausea
1 pt. impaired nasal ventilation
1 pt. Headache
1 pt. Foot and hand paresthesia

The "inverse starving test" was not able to induce a more than 50% reduction of UCB in our patients with GS. We conclude that this test is not a useful provocation test for GS. As previously reported using intravenous protocols for the nicotinic acid test, the oral ingestion of 170 mg nicotinic acid failed to induce an UCB increase > 50% in 3 of our 18 test persons with GS, which results in a sensitivity of only 83%. Confirming previous findings, this UCB increase was less intensive with the oral than with the intravenous approach, respectively [10, 11, 18–20].

One potential cause of the failure of our procedure ist, that the inverse starving test was started after only one overnight fast and a number of bilirubin baseline levels were just above normal. In the classic caloric restriction test, h owever, best specificity and sensitivity was achieved after 48 hours [21–23]. It might be speculated therefore, that 48 hours of restricted calorie intake might induce higher baseline bilirubin levels and improve the results of the inverse starving test. However, it was not our aim to introduce another cumbersome and unpleasant procedure to investigate patients for the GS.

Due to a lack of international standards in the evaluation of patients with UCB elevations, patients usually undergo very different investigations to find out the diagnosis of Gilbert's syndrome. In our cohort, patients who consulted their physicians with jaundice were significantly more likely to undergo invasive diagnostic procedures than patients with an incidental finding of elevated UCB. As the median UCB did not differ in these groups, the doctor's decision for an invasive approach seems to be driven by the patient's demand for a diagnosis but not by a substantiated scientific approach.

In conclusion, the inverse starving test is not an appropriate provocation test for patients with suspected GS. Today, no international gold standard exists for the diagnostic criteria of GS and protocols for the nicotinic acid test are differing in a broad range of its test conditions. Due to its unsatisfying sensitivity and its considerable adverse effects, the nicotinic acid test seems not to be mandatory for the GS diagnosis. A growing number of authors suggest that there is no need for provocation tests at all, if no change in the typical laboratory constellation of an isolated UCB elevation occurred after 3–6 months. In agreement with this, we suggest that the clinical diagnosis of GS can be established in patients with a high fraction of UCB, normal values of liver enzymes and no laboratory signs of haemolysis at the time of the initial consultation and three months later.