Biochemical determinations

Blood determinations: The length of fasting before taking the blood sample was at least 11 hours (Method folder 3.23). Blood was taken for serum-, heparin plasma- and EDTA-sample in 4 ml tubes. 

  • B-Hkr: Haematocrit was determined from the EDTA-blood samples in the field laboratory. The microcapillaries were centrifuged for five minutes at a speed of 12 500 rpm (Adams Autocrit Centrifuge, ser. no. AD 1601).  

The serum and plasma samples were frozen (-20oC) and sent to SII:s laboratory in Turku. The following determinations were made for all participants: 

  • S-Latex and S-Waaler-Rose: Reumafactor was determined at the laboratory at the National Public Health Institute (Aho et al. 1988 and 1989, Heliövaara et al. 1995) (Method folder 3.38). 
  • fS-T4: Thyroxine was determined radioimmunologically using a commercial kit (Lääke Oy). 
  • fS-Chol, fS-HLD-Chol: Serum total cholesterol was determined using a modification of the direct Liebermann-Burchard-method without the so called serum-blanc subtraction (Carr and Drekter 1956). In the HDL-cholesterol determinations, LDL and VLDL were precipitated with Mg-dextranesulphate, and from the supernatant HDL-cholesterol was determined in the same way as described in the total cholesterol method (Kostner 1976, Finley et al. 1978).
  • fS-Trigly: Serum triglycerides were determined using an entirely enzymatic method (Wahlefeld 1974), (Boehringer, Mannheim). 
  • fS-GT: Serumin gammaglutamyltransferase was determined kinetically by measuring p-nitroaniline released from gamma-glutamyl-p-nitroanilid in a glysylglysinibuffer at pH 8.2 (Szasz 1969, Rosalki and Tarlow 1974). 
  • fS-Asat, fS-Afos, fS-Afosis1, fS-Afosis3: Liver enzymes were determined using the methods suggested by the Scandinavian Committee on Enzymes (The Committee on Enzymes of the Scandinavian Society for Clinical Chemistry and Clinical Physiology 1974, Hörder et al. 1981).
  • fP-Gluk: Plasma glucose was determined using a commercial (Boehringer, Mannheim) glucose oxidase method (Werner et al. 1970).
  • fP-Krea: Plasma creatinine was determined using a kinetic modification of the picric acid method (Bartels et at. 1972).

The following determinations were made for a 20% random sample of the participants in the basic health examination and for other specific subgroups:

  • fS-Uraate: Serum urate was determined using the uricase/catalyse method (Kageyama 1971, Thefeld et al. 1973). The reagents were commercial kits (Boehringer, Mannheim R). 
  • fP-K, fP-Na: Plasma kalium and natrium were determined using flame photometry (Corning Flame Photometer 430). The instrument automatically dilutes the sample to 1:200. Litium was an internal standard. 
  • fS-Lipoprotein: The lipoprotein determinations were carried out in the scientific laboratory of III internal medicine clinic of Meilahti hospital using a preparative ultracentrifuge analysis (Aromaa et al. 1985). The fractioning (VLDL, LDL, HDL) with the ultracentrifuge (Havel et al. 1955) was performed using the Beckmann – Spinco L-50 device. From each separated fraction and in addition from the serum sample before centrifugation cholesterol was determined with an enzymatic method (Röschlau et al. 1974) and triglycerides using the Kessler and Lederer (1966) method applied to a Technicon Autoanalyzer.   
  • Apoprotein A1- and A2- determinations were carried out in the scientific laboratory of III internal medicine clinic of Meilahti hospital using an immunodiffusion method (Cheung and Albers 1977).
  • fS-T3, fS-TSH, T3U: Thyroid gland hormone determinations were made using radioimmunological methods based on prepared kits, T3 (Lääke Oy), TSH (Corning), T3U (Amersham).
  • fS-TIBC, fS-Fe, fS-UIBC: Iron and iron binding capacity were determined using a ferrozine method. 
  • Serum digitalis concentration i.e. S-Digoksin and S-Digitoksin were determined for individuals using digitalis with a radioimmunological method (Smith et al. 1969, Aromaa et al. 1985, Impivaara 1986). The determinations were carried out in the department of biomedicine of Turku University. 

For all participants of the basic health examination, the following determinations were made several years later based on a stored frozen serum sample: 

  • Vitamin D (S-25-OH-D): Serum 25-hydroxyvitamin D concentration was determined using radioimmunoassay (RIA, DiaSorin, Stillwater, Minnesota) (Mattila et al. 2007, Knekt et al. 2010)
  • Sensitive CRP (S-C-reactive protein): Walkon CRP-UL, Latex turbidimetricimmunoassay, detection limit 0.06 mg/L.
  • Cotinine and thiocyanate: Serum cotinine concentration was determined using the Nicotine Metabolite radioimmunoassay kit (Diagnostic Products Corporation, Los Angeles, CA, USA) and serum thiocyanate was determined with a colorimetrical method (Korpilähde et al. 2004, Vasankari et al. 2011).
  • Serum insulin was measured using a modification of Herbert’s immunoasay method. Microparticle enzyme immunoassay (MEIA), Insulin kit (REAGENT): Abbott Laboratories, Dainabot, Tokyo, Japan Laite (ANALYZER): ABBOT, IMX 20238. 
  • Tissue transglutaminase antibodies indicating coeliac disease were determined from all serum samples using a commercial kit. As the positivity cut-off point 7.0 units/ml was used (EU-tTG, umana IGA, Eurospital S.p.A., Trieste, Italy). If the result was positive, tissue transglutaminase antibodies with another kit (Celikey tTG, Celikey, Phada, Freiburg, Germany; positivity cut-off point 5.0 units) and also endomysial antibodies (EMA) with an indirect immunofluorescence method (Sulkanen et al. 1998, Lohi et al. 2007). 
  • Urinary determinations: For all participants the following urinary determinations were made: U-Prot-kvl, U-Gluk-kvl, U-Hb-kvl. For women and 50-years old and older men bacterium growth was determined using an Uricult-sheet. Furthermore, bacterium cultivation with sensitivity determination was performed in case the result was positive (Uricult > 104). From 30-59 years old men an overnight urine sample was collected and nU-Krea, nU-Na, nU-K and nU-Rel.den. were determined from it. The methods were the same as in the corresponding serum determinations. The participants were advised not to urinate at least 6 hours before attending the examination.

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