This study was performed according to the Helsinki Declaration and was approved by the Regional Ethics Review Board of Lund University. All subjects gave their written, informed consent before entering the study.
The inclusion criterion of the study was diabetes mellitus in patients above 18 years of age. The first patient at each consultation occasion who fulfilled this criterion when scheduled for routine clinical follow-up at the Department of Endocrinology, Skåne University Hospital, Malmö, from January 2008–February 2010, was invited to participate in the study. Exclusion criteria were severe renal failure demanding dialysis or severe cardiac diseases. At the time of the inclusion, they completed an established questionnaire with 15 symptoms related to disturbances of the gastrointestinal tract (loss of appetite, swallowing disturbances, meal-related cough, early satiety, nausea, vomiting, weight loss, abdominal fullness, bloating, regurgitation, constipation, diarrhea, evacuation incontinence, symptomatic postprandial hypoglycemia and postprandial perspiration), previously used for these patients [2–4]. Types and duration of diabetes mellitus, presence of diabetes complications such as retinopathy (based on fundus photography), angiopathy, microalbuminuria (measured as the albumin/creatinin quotient), albuminuria, peripheral neuropathy (examined by patellar and achilles tendon reflexes, sensitivity to vibration and monofilament test), autonomic neuropathy according to established clinical criteria (sexual dysfunction, profound sweating and orthostatic blood pressure), drug treatments, concomitant diseases, and body mass index (BMI) were all recorded by the physicians.
Glycosylated hemoglobin (HbA1c) was analyzed at the Department of Chemistry, according to clinical routines. HbA1c values were collected as Mono-S and subsequently converted to the National Glycohemoglobin Standardization Program (NGSP) standard by use of the following algorithm: 0.923 x HbA1c (Mono-S) × 1.345 = HbA1c (NGSP) . Percentage HbA1c values were converted to the International Federation of Clinical Chemistry (IFCC) standard in mmol/mol according to the following equation: IFCC (mmol/mol) = ([NGSP (%)] - 2,152)/0.09148 .
The patients were referred to investigations by esophageal manometry and gastric emptying scintigraphy. If both examinations could not be performed, the patient was excluded from the study. Patients were also referred to esophagogastroduodenoscopy when clinically indicated.
Thirty-nine patients of the 55 invited completed the study and provided blood samples for analysis of GnRH antibodies, and were thus finally included in the evaluation. The most common reason not to complete the study was an inability to swallow the manometry catheter.
Standardized esophageal manometry was performed with an intraluminal, solid-state transducer system (Gaeltec Ltd, Isle of Skye, Scotland). Polygraph ID converter digitized the analog signal. The software was PolyGram NET (Medtronic-SynMed Medical, Stockholm, Sweden). All pressure values were expressed in mmHg and referred to atmospheric pressure. The manometry catheter was introduced through the nose and fluoroscopically positioned in the distal esophagus with the patient sitting in an upright position, which is the standard method at our laboratory. With the catheter in place, all participants were instructed to swallow 10 ml of a barium contrast medium (60% w/v). At least five barium swallows were recorded. The video fluoroscopic image and the manometry registration were mixed using a video output card (Medtronic-SynMed Medical) [2, 3]. Patients who fulfilled one or more of the following five criteria in the esophageal manometry with abnormal results were considered to suffer from esophageal dysmotility: 1: Absence of peristaltic contraction in the esophagus (aperistalsis > 0%), 2: Mean peristaltic contraction amplitude < 30 or > 200 mmHg in the esophagus, 3: Percentage of simultaneous, non-propulsive peristaltic waves in the esophagus > 10%, 4: Speed of the peristaltic wave < 3 or > 6 cm/s in the distal esophagus, and 5: Resting pressure in the lower esophageal sphincter (LES) < 10 or > 30 mmHg. Normal peristaltic activity was defined as propulsive contraction waves with peak amplitudes between 30–200 mmHg and a speed between 3–6 cm/s [2, 3, 19, 20].
Gastric emptying scintigraphy
A test meal was prepared by adding tin colloid labeled with 30–50 Mbq of technetium-99 to an egg, which was whipped in a glass cup in a hot water bath until coagulated. The egg and a slice of toasted white bread were cut into pieces smaller than 1 cm × 1 cm and served with 100 ml water at 37°C. The meal was eaten within 5 min. Immediately thereafter, a large-field, double-headed gamma camera (Philips Skylight, Philips Medical Systems, Best, The Netherlands) was placed anteriorly and posteriorly parallel to the upper abdominal wall. The radioactivity was measured continuously (1-min frames) for 70 min starting immediately after meal ingestion. A Region of Interest (ROI) representing the stomach was created and the activity of the first frame was taken as 100%. The gradual decreasing radioactivity, measured as the number of radioactivity decays per minute (counts/min), was plotted against time. The time elapsed to reach a 50% decrease of the activity in the ROI (T50) was identified as the point at which this plot crossed the 50% value. The values of the radioactivity measured were corrected for the half-life of 99mTc, and for attenuation by using the geometrical mean values of the decay curves obtained from the two gamma camera heads used. T50 > 2 standard deviations (sd) for healthy control subjects (70 min) was considered abnormal .
Measurement of human antibodies against gonadotropin-releasing hormone
Blood samples were drawn from patients and the serum was separated and kept frozen at -20°C until analyzed. Analysis of anti-GnRH antibodies was carried out by an ELISA slightly modified on the basis of the results described in previous studies [8, 16]. The wells of micro titer plates were coated with human GnRH (L7134, Sigma, St Louis, MO, USA) for an overnight incubation at 4°C and thereafter the plastic wells were blocked with 0.5% fish gel solution (G7765, Sigma) in PBS containing 0.05% Tween-20 (PBS-T). Serial dilutions of patient serum (1/100, 1/500 and 1/2500 in PBS-T) were then added to the plates and incubated for 2 h at room temperature (RT) and overnight at 4°C. After rinsing with PBS-T, deposition of autoantibodies directed to GnRH was detected using biotinylated rabbit anti-human IgM (673211, MP Biomedicals, Solon, OH, USA) or IgG antibodies (ab7159, ABcam, Cambridge, MA, USA) appropriately diluted in PBS-T. After another incubation for 2 h at RT, the plates were washed and the bound, biotinylated antibodies detected by alkaline phosphatase-conjugated streptavidin (405211, Biolegend, San Diego, CA, USA), incubated for 1 h at RT. To develop a color reaction a phosphatase substrate kit (37620, Pierce, Rockford, Ill, USA) was used. The absorbance at 405 nm was measured after 2 h of incubation at RT. A plasma pool from healthy blood donors was included on each ELISA plate for measurements of the variation. The plasma pool was used for the calculation of the intra-assay and inter-assay coefficient of variation, which was 11.5% and 16.1%, respectively, for IgM and 11.5% and 25.4%, respectively, for IgG. Antibody levels are presented as relative units (RU) (absorbance values after subtraction of background levels and multiplied by 100). Relative units over 0 were considered as a positive antibody level .
The controls were chosen from a cohort of healthy blood donors previously described in detail . Over a period of five months (October 1996–February 1997), blood donors were offered antibody screening for gastrointestinal diseases. To be able to include all blood donors in Malmo, sera from male donors were collected over a 3-month period and from female donors over a 4-month period (in accordance with their regular donation intervals). A total of 1,970 donors were included. During this period, 2,135 blood donations took place, which means that at least 92% of donors agreed to be included. From this sample cohort, 50 men and 50 women from each 10-year age span period, between 20 and 70 years of age, were randomly included. As few blood donors above 60 years of age, only 16 women and 40 men were included in the age group 60–70 years. In total, 456 controls were examined during the same time period as the patients. From this cohort, two age- and gender-matched controls were randomly extracted for each patient in this study.
The data were analyzed using the statistical software package SPSS for Windows© (Release 20.0; IBM). All variables were analyzed for normal distribution by Kolmogorov-Smirnov test. Group-wise differences between patients and controls were tested by using the unpaired Student’s t-test and, when normality was rejected (antibody levels), the Mann-Whitney U-test was used. Fisher’s exact test was used for dichotomized variables. Correlations were calculated by Spearman rank correlation test. Values are expressed as mean ± standard deviation (SD) or median, interquartile range (IQR). Binary logistic regression analysis was performed to test for an association between the presence of antibodies against GnRH (dependent variable) and clinical findings in patients. Independent variables were age (years), gender, type of diabetes, duration of disease (years), BMI (kg/m2), HbA1c (mmol/mol), esophageal and gastric dysmotility, secondary complications, and symptoms, examined as univariate analysis. No presence of antibodies, symptoms or pathological findings was used as reference. P < 0.05 was considered statistically significant.