Study population and design
Figure 1 shows a flow chart of eligible patients, dropouts, and reasons for dropping out of the study. Selection criteria were: (1) men and women aged 30–87 years and (2) no evidence of previous or ongoing neuromuscular disorders. Participants in both groups were selected from the 6th Tromsø Study, a longitudinal population-based study that started in 1974. Novel participants and stratified groups from the 4th Tromsø study; a 10% random sample from age groups 30–39, all participants aged 40–42 and 60–87, and a 40% random sample of subjects aged 43–59 years were invited and data continuously collected from 1 October 2007 to 19 September 2008 in 12,984 participants11. The majority were Caucasians; 87.3% Norwegians, 1.6% Sami, 1.3% Finnish, 2.2% of other ethnicities and 7.6% without information about ethnicity12.
Flowchart of participant inclusion in persistent hyperCKemia and controls.
Participants in the case control study were recruited from the Tromsø study where CK was analyzed in 12,828 subjects. After performing a standardized controlled CK test and matching the groups by age and sex, 113 subjects with persistent hyperCKemia and 128 with persistent normal CK were included in the case–control study. All were Caucasians. During clinical examination, 8 participants were excluded due to previous or ongoing neuromuscular disorders: polyneuropathy (n = 4) including one with Churg-Strauss syndrome, polymyositis (n = 1), myasthenia gravis (n = 1), congenital polio (n = 1), facioscapulohumeral muscular dystrophy (n = 1) (Fig. 1). In total, one with polyneuropathy was excluded from the control group and 7 from the case group.
We used the Scandinavian NORIP references to define hyperCKemia (men < 50 years: 50–400 U/L, men ≥ 50 years: 40–280 U/L and women: 35–210 U/L)13. A total of 686/12,984 (5.3%) subjects with high CK were invited to a standardized control test after being instructed to refrain from muscle strain, trauma, physiotherapy, acupuncture, and alcohol intake 3 days prior to the CK control test. Persistent hyperCKemia was diagnosed in 169 subjects (Fig. 1). Age- and sex matched controls within 30–50 percentile of the CK references from the sample of participants with normal CK were selected and matched if the ages were within 5-year group intervals, and then recruited to the case control study. A study consultant (AKK) administered the participant logistics to ensure a double-blind setting throughout the study (i.e., neither the participants nor the examiners were aware of the CK levels).
Clinical and biochemical parameters
The participants were consulted at Department of Neurology and Neurophysiology, University Hospital of North Norway. A comprehensive evaluation was performed to detect information about general health, diseases and risk factors associated with neuropathy and myopathy. This included current smoking, history of coronary heart disease and kidney disease, presence of hypertension, diabetes mellitus, hypothyroidism, use of medication such as lipid lowering drugs, and waist and hip circumference used to calculate waist-hip ratio. Furthermore, background variables associated with elevated CK were selected in accordance with literature recommendations14,15,16,17. Data collection included questionnaires, clinical and neurophysiological examinations, and biological sampling. Additional information and confirmation of medical history reported by the participants were obtained from our hospitals record’s system. Some of the demographic and clinical information used in the study were selected from questionnaires used in the Tromsø Study.
The following parameters were obtained from a structured interview: Ethnicity (“Caucasian”, “African”, “Other”), previous or current peripheral nerve disease and myopathy, cancer, systemic metabolic disease, haematological disorders, thyroid and parathyroid disease and liver disease, malignant hyperthermia or malignant neuroleptic syndrome, physical exercise (mild activity; activity without sweating or breathlessness < 3 h per week, high leisure physical exercise; activity with sweating or breathlessness ≥ 3 h per week, Alcohol Use Disorders Identification Test (AUDIT) where a score ≥ 8 indicates harmful alcohol consumption18, use of medication reported to be associated with elevated CK; lipid lowering drugs (statins)19, betablockers20, clozapine21 and isotretinoin22. Known muscle disease among first and second degree relatives, presence of muscle pain, stiffness or cramps the last two weeks and fatigue severity scale were recorded23.
Height and weight were measured standardized with light clothing without shoes, and body mass index (BMI) calculated as weight (kg) divided by height squared (m2). Blood pressure was measured in supine position by using a digital monitor (A&D Model UA-779; A&D Instruments Ltd, Abingdon, Oxon, UK). Hand grip strength (kPa) was measured in dominant hand by a Martin vigorimeter; Elmed Inc., Addison, IL, USA24. The best of three efforts was recorded. Knee extension of the dominant leg was tested using a Cybex NORM dynamometer (CSMI, Norwood, MA, USA). After a short, standardized warm up, the participants performed 3 subsequent knee extension and flexions. The average Nm of 3 tests were recorded. Medical Research Council (MRC)-sum score ranging from 0 to 60 assessing global muscle strength on both sides25 and a complete clinical neurological examination was performed using Neuropathy Impairment Score (NIS), a clinical neuropathy instrument with a total score ranging from 0 (normal) to 240. Sensation and reflexes were graded from 0 (normal) to 2 (absent) and muscle weakness from 0 (normal nerve function) to 4 (paralyses)26. Total NIS score and numbers/frequencies with distal sensory loss, impaired ankle reflexes and distal muscle weakness were recorded. Clinical examinations were performed by experienced neurologists (HL, SIB).
Serum CK was automatically analyzed within 6 h from sample withdrawals at the Department of Clinical Chemistry, University Hospital of North Norway (CK-NAC, Roche Diagnostics, Mannheim, Germany) with an analytic variation coefficient of ≤ 1.6%. All biochemical tests were analyzed at the same laboratory and included liver transaminases, lactate dehydrogenase, serum lipids, high-sensitive C-reactive protein (hs-CRP), serum creatinine, non-fasting serum glucose, glycated hemoglobin A1c (HbA1c-%), vitamin B12, folate and thyroid hormone levels4,11.
Neurophysiological examinations
Nerve conduction studies (NCS) and electromyography (EMG) were conducted by experienced neurophysiologists (KA, SL) in the neurophysiology lab at Tromsø University Hospital using Keypoint Classic equipment (Medtronic, Skovlunde, Denmark) according to standard techniques27. The NCS examinations were performed unilaterally on the dominant side. Surface electrodes were utilized for motor and sensory stimulation and registration. Motor NCS included the median, ulnar, and tibial nerves, and sensory NCS the median and ulnar nerves (orthodromic stimulation) and the sural nerve (antidromic stimulation). For motor nerves the following parameters were analyzed: distal latency, amplitude, conduction velocity (CV), and minimum F-M wave latency of 20 supramaximal stimuli, and for sensory nerves: distal latencies, amplitudes, and CVs. EMG examinations with concentric needle were performed in extensor digitorum communis, vastus lateralis and anterior tibial muscles unilaterally on the dominant side. Spontaneous activity with muscle at rest was assessed (fibrillation potentials and positive sharp waves in x out of 10 needle positions, fasciculation potentials, complex repetitive discharges, and myotonic discharges). With slight voluntary muscle contraction, 20 unique motor unit potentials (MUPs) were collected, and amplitude, duration and polyphasia (%) were recorded. At last, interference pattern with gradually increasing muscle contraction were recorded (10 different measurements of turns amplitude with references from the manufacturer).
Statistical analysis
Background variables and endpoints were evaluated by inspection of histograms which showed right-sided skewness for NIS and CK. Non-parametric test was therefore used for NIS and CK was analyzed log transformed. Descriptive data are presented as mean and standard deviation (SD), median (IQR) or number and frequency. Student´s t-test, Mann–Whitney U test respectively χ2-test were used to assess statistical differences between means, medians and frequencies of data and Pearson correlation coefficient used to measure strength and direction between continuous variables while Spearman rank correlation was used to measure associations between NIS score and neurophysiological parameters. Variables statistical significantly associated with persistent hyperCKemia were included in logistic regression analyses to adjust for confounders. In cases with colinearity, like ALT and AST, the one with the strongest association was selected. The analyses were performed repeatedly in model 1 (tibial nerve cMAP amplitude) and model 2 (tibial nerve MNCV). Two-sided p < 0.05 was considered statistically significant. All analyses were conducted by SPSS software (Statistical Package for Social Science INC, Chicago, Illinois, USA), version 29.
Ethical declaration
This study was conducted according to the Declaration of Helsinki and was approved by theThe Regional Ethical Committee for research (approval number: REK NORD 11/2008). Informed consent was obtained from all subjects.
Consent to participate
All subjects in the study gave their written consent prior to inclusion in the study.