1760 Diabetes Volume 64, May 2015 Anne-Sophie Sejling,1,2 Troels W. Kjær,3,4,5 Ulrik Pedersen-Bjergaard,2 Sarah S. Diemar,2,4 Christian S.S. Frandsen,4,6 Linda Hilsted,5 Jens Faber,4,7 Jens J. Holst,4 Lise Tarnow,2,8 Martin N. Nielsen,6 Line S. Remvig,9 Birger Thorsteinsson,2,4 and Claus B. Juhl1,9,10 Hypoglycemia-Associated Changes in the Electroencephalogram in Patients With Type 1 Diabetes and Normal Hypoglycemia Awareness or Unawareness Diabetes 2015;64:1760–1769 | DOI: 10.2337/db14-1359 Hypoglycemia is associated with increased activity in during hypoglycemia are not affected by awareness the low-frequency bands in the electroencephalogram status during a single insulin-induced episode with (EEG). We investigated whether hypoglycemia aware- hypoglycemia. ness and unawareness are associated with different hypoglycemia-associated EEG changes in patients with type 1 diabetes. Twenty-four patients participated In type 1 diabetes, the major limiting factor in achieving in the study: 10 with normal hypoglycemia awareness glucose targets is risk of severe hypoglycemia (1). Im- and 14 with hypoglycemia unawareness. The patients paired hypoglycemia awareness (reduced ability to – were studied at normoglycemia (5 6 mmol/L) and hy- perceive the onset of hypoglycemia) is associated with a – – COMPLICATIONS poglycemia (2.0 2.5 mmol/L), and during recovery (5 6 6- to 20-fold increased risk of severe hypoglycemia (2,3). mmol/L) by hyperinsulinemic glucose clamp. During It is assumed that episodes of repeated mild symptom- each 1-h period, EEG, cognitive function, and hypo- atic and asymptomatic hypoglycemia contribute to the glycemia symptom scores were recorded, and the development of impaired hypoglycemia awareness (4,5). counterregulatory hormonal response was measured. Quantitative EEG analysis showed that the absolute The condition is characterized by loss of hypoglycemia amplitude of the u band and a-u band up to doubled warning symptoms and blunted counterregulatory hor- – during hypoglycemia with no difference between the mone responses to low blood glucose (6 8). Thus, the two groups. In the recovery period, the u amplitude threshold at which the patients experience symptoms of remained increased. Cognitive function declined equally hypoglycemia will gradually decrease. For some individ- during hypoglycemia in both groups and during recovery uals, this level is equal to or below the threshold for reaction time was still prolonged in a subset of tests. The neuroglycopenia (9,10). aware group reported higher hypoglycemia symptom The hypoglycemia-associated neuroglycopenia, result- scores and had higher epinephrine and cortisol responses ing in neuroglycopenic symptoms and cognitive dysfunc- compared with the unaware group. In patients with type tion, is mirrored in the electroencephalogram (EEG) by an 1 diabetes, EEG changes and cognitive performance increased activity in the low-frequency bands (u and delta 1Faculty of Health, University of Southern Denmark, Odense, Denmark Corresponding author: Anne-Sophie Sejling, [email protected]. 2 Nordsjællands Hospital Hillerød, Hillerød, Denmark Received 6 September 2014 and accepted 2 December 2014. 3Roskilde Hospital, Roskilde, Denmark Clinical trial reg. no. NCT01337362, clinicaltrials.gov. 4Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark This article contains Supplementary Data online at http://diabetes 5Rigshospitalet, Copenhagen, Denmark .diabetesjournals.org/lookup/suppl/doi:10.2337/db14-1359/-/DC1. 6Hvidovre Hospital, Hvidovre, Denmark © 2015 by the American Diabetes Association. Readers may use this article as 7Herlev Hospital, Herlev, Denmark long as the work is properly cited, the use is educational and not for profit, and 8Health, Aarhus University, Aarhus, Denmark the work is not altered. 9HypoSafe A/S, Lyngby, Denmark 10Sydvestjysk Sygehus, Esbjerg, Denmark diabetes.diabetesjournals.org Sejling and Associates 1761 band) (11). These changes disappear when the glucose concentration is slightly increased, even before normogly- cemia is restored (12,13). This is in contrast to hypoglycemia- induced cognitive dysfunction, which is present up to 75 min after glucose levels are restored (14). While differences in symptomatic and hormonal coun- terregulatory responses between subjects with normal or impaired awareness are well known, less is known about differences in neuroglycopenia as determined by EEG and cognitive evaluation during hypoglycemia and recovery Figure 1—Study design. A hyperinsulinemic euglycemic-hypoglycemic following hypoglycemia. The aim of our study was to glucose clamp was performed to reach each glycemic level. The assess whether hypoglycemia awareness status is associ- normoglycemia part of the study started when the subject’s plasma ated with differences in the hypoglycemia-induced EEG glucose level had decreased to 5 to 6 mmol/L. The subject was examined during normoglycemia, hypoglycemia, and recovery fol- changes during and shortly after an episode of mild, lowing hypoglycemia. EEG was recorded at rest. Symptom scores insulin-induced hypoglycemia in hypoglycemia-aware and were obtained after each EEG recording at rest. Bl t, blood tests for -unaware patients with type 1 diabetes. analysis of the counterregulatory hormones; Cog, cognitive function assessed by CalCAP test, TMT B, and the Stroop test. *Plasma glucose measurement. RESEARCH DESIGN AND METHODS The study was a clinical controlled study. The protocol is registered at http://clinicaltrials.gov (NCT01337362) and was approved by the Regional Committee on Health Re- On the day of the experiment, subjects arrived in the search Ethics. Written informed consent was obtained clinical research unit after an overnight fast. In case of from all participants. glucose measurements ,3.5 mmol/L in the preceding Subjects 24 h, study procedures were postponed 2 weeks. Twenty-four patients with type 1 diabetes were recruited from the diabetes outpatient clinics at Nordsjællands The Hyperinsulinemic Hypoglycemic Clamp Procedure – Hospital Hillerød and Steno Diabetes Center, Denmark. The glycemic targets were 5 6 mmol/L during normogly- – Inclusion criteria were type 1 diabetes for .5years,age cemia, 2.0 2.5 mmol/L during hypoglycemia (nadir 2.2), – .18 years, and being either hypoglycemia aware or un- and 5 6 mmol/L during recovery (Fig. 1). For the clamp aware. Exclusion criteria included pregnancy; breastfeed- procedure, insulin (Actrapid; Novo Nordisk, Ballerup, ing; any brain disorder; use of antiepileptic drugs, Denmark) mixed with heparinized plasma from the pa- b-blocking drugs, or neuroleptic drugs; use of benzodia- tient and isotonic saline was administered intravenously zepines within the last month; cardiovascular disease; at a rate of 1 mU insulin/kg/min. A variable 20% glucose and alcohol or drug abuse. Hypoglycemia awareness sta- infusion was administered to keep plasma glucose at the tus was classified by the Pedersen-Bjergaard method desired levels. (15), the Gold score (16), andtheClarkemethod(17). EEG at Rest Of the 24 participants, 14 patients were classified as Digital EEG was measured continuously (Cadwell, Kenne- hypoglycemia unaware and 10 patients as hypoglyce- wick, WA). The electrodes were placed according to the mia aware according to all three methods (Supplemen- 10–20 system using electrocaps. Data were collected at tary Table 1). Patients who did not qualify as either a sampling rate of 200 Hz and filtered using a first-order hypoglycemia aware or unaware were excluded from high-pass filter of 0.5 Hz and a first-order low-pass filter participation. at 70 Hz. Explicit care was taken to obtain 5 min of Experimental Protocol electroencephalographic standard conditions with eyes From 5 days before the hypoglycemic clamp, the closed during two specific time points at each glycemic participants wore a continuous glucose monitor (CGM) level subsequently analyzed in tandem. Results from P3- (Guardian Real-Time with Enlite sensor; Medtronic, C3 electrodes are reported. This location in the parieto- Minneapolis, MN) to detect any hypoglycemia in the central brain region was chosen because hypoglycemia- days before the experiment. In order to reduce the risk of associated EEG changes are most abundant in this hypoglycemia, the CGM was set to alarm the participant if area (18). glucose levels fell ,4.5 mmol/L. This was done to com- Analyses of the EEG were performed by quantitative pensate for possible inaccuracies and to allow the patient EEG (qEEG) analysis focusing on frequency characteristics time to take corrective measures before the glucose of the data in the 1) u band (4–7.75 Hz), where activity is concentration fell ,3.5 mmol/L. All patients used the associated with drowsiness, mediation, or light sleeping; Contour Link blood glucose meter (Bayer HealthCare, 2) a band (8–12.75 Hz), where activity is associated with Leverkusen, Germany) for blood glucose monitoring and relaxation with eyes closed; and 3) a combined a-u band calibration of the CGM. (4–12.75 Hz). The a-u band was included in order to 1762 EEG and Hypoglycemia Awareness or Unawareness Diabetes Volume 64, May 2015 detect whether any shift in frequency occurred in the measurement, and glucagon was measured with a radio- transition between the a and u band that might not oth- immunoassay directed against the COOH terminus of the erwise be identified. Power spectral density of the normo- glucagon molecule (antibody code number 4305). Serum glycemic, hypoglycemic, and recovery periods was estimated