We conducted a randomised, double-blind, single-centre, two-stage, crossover trial to compare the hypoglycaemic responses to single doses of insulin detemir and NPH insulin in participants with type 1 diabetes. Participants were recruited from the Royal Surrey County Hospital diabetes database. All gave written informed consent and the local Ethics Committee approved the study. Participants were excluded if there were factors that would alter sensitivity to hypoglycaemia, including previous reduced awareness of hypoglycaemia, autonomic neuropathy, treatment with beta-blockers and a total daily insulin dose greater than 100 IU. At initial screening, plasma HbA1c and C-peptide concentrations, full blood count, and renal and liver function were measured.
We studied 18 participants, of whom 14 completed both arms of the study satisfactorily; four participants were excluded from the final analysis before it was unblinded, due to failure to achieve hypoglycaemia on both occasions. Height and weight were measured at screening and before each study. The screening weight was used for calculation of the insulin dose administered on each study day.
Participants were asked not to undertake vigorous physical activity and not to drink alcohol, and were excluded if they reported an episode of hypoglycaemia in the 72 h before attendance. Injection of basal insulin was omitted from the evening before participants attended for an overnight variable infusion of soluble (human) insulin to achieve a stable basal plasma glucose concentration of approximately 7 mmol/l by the following morning. This protocol was chosen to ensure that overnight hypoglycaemia was avoided and to enable a washout period of a minimum 34 h prior to subcutaneous injection of the trial insulin. Participants then underwent two studies, one with insulin detemir, the other with NPH insulin, in random order with an interval between studies of 7 to 42 days. Intravenous access was obtained via both ante-cubital fossae, enabling sampling from one and infusion into the other. During the study participants remained fasting, were allowed to ingest water only and were supine throughout. After drawing baseline (unenriched) blood samples, a primed constant infusion of [6, 6-2H2]glucose (170 mg, 1.7 mg/min) was started. A period of 160 min was allowed for isotopic equilibration, prior to the subcutaneous injection of insulin. The isotope infusion was then continued until the end of the study.
Insulin detemir or NPH 0.5 U/kg was injected subcutaneously into a thigh in a double blind manner by a diabetes specialist nurse not otherwise involved in the trial. This dose was chosen after a pilot study of five participants dosed at 0.4 U/kg failed to achieve adequate hypoglycaemia. An even higher dose might have led to an earlier nadir of blood glucose, but symptoms of hypoglycaemia might have been less tolerable, leading to withdrawal of a larger number of participants. The rate of infusion of intravenous human insulin was gradually reduced to zero over the following 10 min.
The protocol was divided into three sections according to the glucose concentration. Section 1 began with glucose stabilisation prior to injection and lasted until blood glucose had fallen to 4 mmol/l. Section 2 was the period of glucose decline from 4 to 2.4 mmol/l, ending either then or when the symptoms of hypoglycaemia became intolerable or after 8 h from injection. Section 3 was the rescue period, when 20% dextrose primed with [6, 6-2H2] glucose was infused at 6 mg/kg to restore blood glucose to 6 mmol/l.
Blood glucose concentration was monitored by frequent blood sampling initially at 20 min intervals, increasing when blood glucose had fallen below 4 mmol/l to 5 to 10 min intervals. Samples were taken for enriched glucose and plasma glucose concentrations every 20 min throughout. Insulin detemir or human insulin concentration was measured every hour throughout the study.
Initially, the counter-regulatory hormones (plasma adrenaline [epinephrine], noradrenaline [norepinephrine], glucagon, serum growth hormone, cortisol and NEFA) were measured every hour. When the blood glucose concentration was at or below 4 mmol/l, these were measured at 20 min intervals until hypoglycaemia nadir, when they were taken just before initiation of the rescue infusion, and at 10 and 30 min thereafter. The target glucose level for termination of the study was 2.4 mmol/l. If this value was not reached within 8 h after subcutaneous injection of the trial insulin, the study was ended. Some participants reached a plateau above 2.4 mmol/l. The study was then stopped at the time limit of 8 h or at a time when participants could no longer tolerate the symptoms of hypoglycaemia. Once the rescue infusion of 20% dextrose had restored blood glucose to 6 mmol/l, food and drink were given.
Cognitive function tests
The cognitive function tests were performed at baseline (−60, −30, −15 min) and then hourly after subcutaneous insulin administration until blood glucose reached 4 mmol/l. Thereafter, the tests were performed every 15 min.
Finger tapping test
A computer key was pressed as many times as possible for 10 s. The test was repeated three times at each defined time-point and the mean calculated .
Four-choice reaction time test
A computer screen randomly displayed a dot in one of four possible fields. The participant had to respond by pressing the correspondingly positioned key on a keypad. Each test was carried out with 100 cycles. Number correct (out of 100), mean correct time (ms) and standard deviation for correct time (ms) were recorded .
Hypoglycaemia symptom score
A hypoglycaemia symptoms questionnaire based on the Edinburgh Hypoglycaemia Scale  with a linear interval scale for each symptom was completed. At each stage the question was asked ‘Do you feel hypo?’ and hypoglycaemia symptom scores (HSS) for autonomic symptoms, neuroglycopenic symptoms and general malaise were graded from 1 (not at all) to 7 (a great deal). This was completed at baseline (−180 min) and hourly after insulin administration until glucose concentration reached 4 mmol/l. After this, the questionnaire was completed every 15 min.
Heart rate and blood pressure were recorded at baseline and every 30 min until blood glucose concentration reached 4 mmol/l. From then they were recorded every 15 min until blood glucose concentration was restored.
The total concentrations of free and bound insulin detemir in serum were measured with an insulin detemir-specific ELISA . Concentrations of serum human insulin and C-peptide were analysed using an insulin ELISA assay (Dako, Glostrup, Denmark).
Plasma concentrations of adrenaline and noradrenaline were quantified by HPLC (Chromosystems, Munich, Germany), glucagon by RIA (Diagnostic Products Corporation, Los Angeles, CA, USA), growth hormone using serum-coated bead immunoradiometric assay (Skybio, Wyboston, UK), NEFA using an enzymatic assay (Randox Laboratories, Crumlin, UK) and cortisol by ELISA (DRG International, Mountainside, NJ, USA).
All isotopic enrichments were measured by gas chromatography–mass spectrometry on a mass selective detector (HP 5971A; Agilent Technologies, Queensferry, UK). The isotopic enrichment of glucose was determined using a penta-O-trimethylsilyl-d-glucose-O-methoxime derivative analysed by selecting ion monitoring of the ions at charge:mass ratio 319 and 321. Glucose concentrations in blood and plasma were measured by a glucose oxidase method using a glucose analyser (Clandon Scientific; Yellow Springs Instruments, Yellow Springs, OH, USA)
Endogenous glucose production (EGP) and peripheral glucose uptake (PGU) were calculated using Steele’s non-steady state equations  adapted by Finegood et al.  and modified for stable isotopes. Before calculation of glucose turnover, plasma glucose concentration and glucose enrichment time courses were smoothed and interpolated to 5 min intervals using optical segments technique analysis . For each time-point EGP and PGU were calculated. For each participant in each study the mean EGP and PGU at a glucose concentration of 7.0, 6.5, 6.0, 5.5, 5.0, 4.5, 4.0, 3.5, 3.0 and 2.5 mmol/l were calculated. The boundaries for each concentration were ±0.05 mmol/l, so for example, any concentration between 5.95 and 6.05 mmol/l was considered to be a concentration of 6 mmol/l. For each concentration there were one, two or three values. The mean EGP and PGU values at each of these glucose concentrations were calculated.
The following were compared: baseline adjusted HSS at nadir blood glucose concentration, hypoglycaemic symptoms, hypoglycaemic awareness, blood glucose profile, autonomic response (time taken for heart rate to be >15% baseline), counter-regulatory hormones, cognitive function, EGP, PGU and the pharmacokinetics of insulin detemir and human insulin.
Data are expressed as mean values with standard deviations or standard errors as appropriate. EGP and PGU were calculated and plotted against blood glucose concentration. Mean profiles for all time-dependent variables were plotted as a function of time using time since trial drug injection and time since blood glucose nadir. A test for no difference in symptoms of hypoglycaemia between insulin detemir and NPH insulin was carried out using ANOVA with insulin preparation, interactions between insulin preparation and dose level and order of visit as fixed effects. A one-way ANOVA was applied to EGP and PGU values for glucose concentrations during hypoglycaemia, (3.5-2.5 mmol/l).