ANNEX I RELATIONSHIPS BETWEEN DOSE AND EFFECTSupplementary provisions
The blood lead levels used in the assessment of the results of the biological screening are based on analysis of the scientific data concerning the various toxic effects of lead. With this analysis, which takes account of normal variations in biological values within the population, it is possible to establish quasi-quantitative relationships between dose and effect. The following relationships between dose and effect are used for references for the purposes of this Directive. A decrease in ALAD activity in the red corpuscles as a result of exposure to lead may be tolerated for the population, provided that it does not interfere with haematopoiesis. For blood lead levels below 15 to 20 ¶g/100 ml, a decrease in ALAD activity is not currently considered to interfere with haematopoiesis. An increase in the protoporphyrin content of erythrocytes in the blood (PPE) indicates interference with the body's use of iron and thus with synthesis of the haem. An increase in PPE has been found at blood lead levels over 20 to 30 ¶g/100 ml. An increase in PPE may however be due to other causes. Interference with the glutathione synthesis may be tolerated only for a small fraction of the population and only if it is slight and does not result in any other sub-clinical symptoms. This unacceptable interference with glutathione synthesis is not found at blood lead levels below 30 ¶g/100 ml. A significant increase in the excretion of delta-aminolevulinic acid in the urine (ALAU) is a significant sign of disturbance in the metabolism of porphyrins and thus a sign of health impairment. A statistically significant increase in ALAU begins to be found at blood levels above 35 ¶g/100 ml. ANNEX II CORRESPONDENCE BETWEEN BLOOD LEAD LEVELS AND ENZYMATIC ACTIVITY For the purposes of Article 2 of this Directive, the relationship between blood lead levels and ALAD enzymatic activity measured in accordance with the European standardized method (Annex III) shall be as follows: >PIC FILE= "T0010805"> If the ALAD values measured are significantly higher than the above limits for the different sections of population, confirmatory blood lead measurements shall not be required. ANNEX III TECHNICAL INSTRUCTIONS FOR MEASURING ALAD ACTIVITY European standardized method for determining the activity of delta-aminolevulinic acid dehydratase The principle of the method adopted to determine the activity of delta-aminolevulinic acid dehydratase is well known. It is based on incubation of the enzyme with an excess of delta-aminolevulinic acid substrate. The porphobilinogen (PBG) which forms after a certain length of time is mixed with the modified Ehrlich reagent and the colour obtained is measured against white using a photometer. The quantity of porphobilinogen produced constitutes a measurement of ALAD activity. METHOD Effect of light Recent experiments have shown that PBG in particular is very sensitive to light. The entire analysis should thus be conducted with no direct sunlight at all in the laboratory (and not simply excluded from the point at which the analysis is carried out). Phase one - Blood sampling and preservation before analysis - Take a sample of 2 ml venous blood using a plastic syringe (not lead stabilized) in the presence of dried heparin ( - Prepare immediately and cool to 4 ºC four samples of 0 72 ml placed in plastic tubes (not lead stabilized). Graduated Marbourg-type pipettes should be used. - If the samples are analyzed within three hours, there is no need to chill them. - Samples should not be preserved at 4 ºC for longer than 24 hours. - Just before analysis, all samples should be placed in a bath of iced water for 10 minutes. Note : Plastics which may be used include, for example, polythene, polystyrene and polypropylene. The preservation period of 24 hours at 4 ºC is a cautious estimate. This interval is long enough for the sample to be removed for analysis from the point at which it was taken to a central laboratory. Three of the four blood samples are used to measure ALAD and the fourth as a blank. Phase two - Haematocrit reading This reading must be taken: - at the same time as the blood sample, - by a capillary method using two samples. The sample should be centrifuged, after closing one end of the tube, at speed of a least 30 000 rev/minute and for not less than five minutes. Note : This reading should preferably be taken at once, but in any case not later than 24 hours after the blood sample is taken. If possible a microhaematocrit centrifuge should be used. Phase three - Haemolysis - Take three blood samples which have been "thawed" using 1 73 ml distilled water (preheated to 37 ºC) and haemolyse for 10 minutes at 37 ± 0 72 ºC. - Use, for preference, a 2 ml graduated pipette for adding the water and then mix thoroughly. - Do not shake the samples at this stage. Note : It has been decided that water should be used rather than Triton X 100 for the haemolysis. Haemolysis experiments using Triton X 100 resulted in a considerable slowing in ALAD activity which has not yet been accounted for and which might be an artifact. Phase four - Addition of the ALA solution to the haemolysate - Prepare the ALA solution. - It should not be prepared more than five hours beforehand. - Heat this solution to 37 ºC for at least 10 minutes before adding it. - Add 1 ml of this solution to the haemolysate, preferably using a 1 ml volumetric pipette, and mix. Note : A pH value of 6 74 has been taken, since experiments have shown that it is at this pH that there is the best correlation between ALAD activity and blood lead level in normal populations. It is not necessary to obtain the maximum activity (which can be done by raising the pH), since the activities to be measured are already fairly high. Phase five - Preparation of the blank - Take 0 72 ml of blood treated as when measuring ALAD up to the point where the ALA solution is added ; in place of this, add 1 ml of TCA-HgCl2 solution, 1 ml of ALA solution, and then proceed as when measuring ALAD (see below). - Add the solutions using volumetric pipettes. Note : Only one blank is compared against a series of three tests for each blood sample. If the OD obtained for the blank is very high, the operation should be repeated to check it. Phase six - Incubation - 60 minutes at 37 ± 0 72 ºC in a water bath. - Incubation time runs from addition of the ALA solution. Note : The incubation period of 60 minutes was chosen in order to increase ALAD activity by natural means, given that no other phase leads to any artificial increase in activity. Experiments have shown that the incubation time/activity ratio is linear for intervals over two hours. The incubation temperature has been kept at 37 ºC for practical reasons. Phase seven - Halting the PBG reaction - Add 1 ml of TCA-HgCl2 solution to the incubation mixture, preferably using a 1 ml volumetric pipette. Phase eight - Centrifugation and filtration - 30 000 rev/minute. - Filtration using Whatman No 54 paper or equivalent (acid-resistant). Note : Centrifugation should last about 10 minutes. The filtration phase is included in order to avoid the transfer by pipette of small particles on to the surface of the supernatant liquid. These particles seem to produce a colour reaction with Ehrlich's reagent. A number of tests have shown that inclusion of the filtration phase leads to better reproducibility. This phase can if necessary be replaced by a second centrifugation. Phase nine - Reaction with Ehrlich's reagent - Mix 1 ml supernatant liquid with 1 ml modified Ehrlich's reagent, using a 1 ml volumetric pipette. - Use a Vortex-type mixer to ensure that the mixture is homogeneous. - Allow the reaction to continue for five minutes before measuring extinction. Note : To obtain a homogeneous mixture it is very important to ensure that the filtered supernatant liquid is well mixed with the Ehrlich's reagent. Phase ten - Extinction measurement - Calibrate the spectrophotometer using a phenolphthaline solution in a basic buffer. - Compare the extinction measurement of the sample with that of the blank at 555 nm in a 1 cm cell (or 2 cm if the absorption is very low). Phase eleven - Calculation of enzyme activity - The equation for calculating enzyme activity is as follows: >PIC FILE= "T0010806"> where: OD = extinction measured, 60 = incubation time, 35 = dilution factor, 62 = molal extinction coefficient in cm2/¶mol, K = spectrophotometric correction coefficient. Note : The units proposed are consistent with the recommendations of the International Union of Biochemistry on enzyme nomenclature. SOLUTIONS 1. Preparation of an ALA solution Solution A: 1 778 g Na2HPO4 7 2H2O dissolved in 100 ml distilled water (preferably deionized). Solution B: 1 738 g NaH2PO4 7 1H2O dissolved in 100 ml distilled water. 29 ml of solution A + 71 ml of solution B give a buffer of 0 71 M sodium phosphate at pH 6 74. This buffer strength is necessary to prevent any fluctuation of pH value in the solution undergoing the reaction. 167 76 mg ALA-HCl are dissolved in solution B (which must always be acid) ; the pH is adjusted to 6 74 on the basis of solution A. The volume is then made up to 100 ml using the buffer solution of 0 71 M sodium phosphate at pH 6 74. This preparation gives a solution of 0 701 M ALA. 2. TCA-HgCl2 solution 1 735 g HgCl2 are dissolved in 100 ml 10 % trichloracetic acid. 3. Ehrlich's reagent solution Reagents: 2 75 g p-dimethylaminobenzaldehyde (pDMAB), 0 725 g HgCl2 dissolved in 10 ml glacial acetic acid, perchloric acid SG 1 77, glacial acetic acid. Preparation: Dissolve the pDMAB in 50 ml acetic acid. Add 24 75 ml perchloric acid and 4 ml HgCl2 solution. Mix, cool and make up to 100 ml with the glacial acetic acid in a graduated flask (1). Store in a dark bottle. Calibration: Community-level calibration should be carried out every year by a laboratory jointly appointed by the Member States. (1)If a brown coloration appears at this point, the reagent should be discarded.