Supplementary Figure 1. A correlation matrix of all the Finnish study cohorts with pairwise Spearman s rank correlation. Only correlations with

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1 Supplementary Figure 1. A correlation matrix of all the Finnish study cohorts with pairwise Spearman s rank correlation. Only correlations with P<0.05 are plotted. Metabolite abbreviations are stated in Supplementary Table 1.

2 Supplementary Figure 2 a-d. Heat maps demonstrating age-related changes in the metabolites in 3,450 FINRISK-97 men (a-b) and 3,009 women (c-d). Figures a and c allow direct comparison with the Estonian data in Supplementary Fig. 3. Figures b and d present the metabolites only measured in the Finnish participants. One-year age groups are displayed on the y-axis and individual metabolites on the x-axis. Colors represent the divergence in units of standard deviation from the mean of the metabolite levels of year-olds. The horizontal line is set at the mean age of menopause, 51 years. Abbreviations of metabolites are explained in Supplementary Table 1. *Individuals aged 73 and 74 are combined in the last age group. Supplementary Figure 2a. Metabolite changes by age in 3,450 FINRISK-97 men.

3 Supplementary Figure 2b. Metabolite changes by age in 3,450 FINRISK-97 men.

4 Supplementary Figure 2c. Metabolite changes by age in 3,009 FINRISK-97 women.

5 Supplementary Figure 2d. Metabolite changes by age in 3,009 FINRISK-97 women.

6 Supplementary Figure 3 a-b. Heat maps demonstrating age-related changes in the metabolites in the Estonian men (a, N=3,542) and women (b, N=6,416). Oneyear age groups are displayed on the y-axis and individual metabolites on the x-axis. Colors represent the divergence in units of standard deviation from the mean of the metabolite levels of year-olds. The horizontal line is set at the mean age of menopause, 51 years. Abbreviations of metabolites are explained in Supplementary Table 1. *Individuals aged >80 are combined in the last age group. Supplementary Figure 3a. Metabolite changes by age in 3,542 Estonian men.

7 Supplementary Figure 3b. Metabolite changes by age in 6,416 Estonian women.

Supplementary Figure 4 a-h. Effect of age on standard deviation standardized metabolite values in the Finnish study populations (n=16,107, of which 7,786 men and 8,321 women).

8 Supplementary Figure 4 a-h. Effect of age on standard deviation standardized metabolite values in the Finnish study populations (n=16,107, of which 7,786 men and 8,321 women). Natural cubic regression splines were utilized with knots placed every 5 years. Women are plotted with a red continuous line and men with a blue dashed line. VLDL=Very low density lipoprotein, LDL=Low-density lipoprotein, HDL=High-density lipoprotein. Supplementary Figure 4a. Age effects on VLDL-related measures presented with natural splines.

9 Supplementary Figure 4b. Age effects on LDL-related measures presented with natural splines.

10 Supplementary Figure 4c. Age effects on HDL-related measures presented with natural splines.

11 Supplementary Figure 4d.Age effects on other lipid measures presented with natural splines.

12 Supplementary Figure 4e. Age effects on amino acids presented with natural splines.

13 Supplementary Figure 4f. Age effects on fatty acids presented with natural splines.

14 Supplementary Figure 4g. Age effects on fatty acids and related measures presented with natural splines.

15 Supplementary Figure 4h. Age effects on small molecules presented with natural splines.

16 Supplementary Table 1. The metabolites studied and their classification. Metabolite group Lipoproteins Metabolite subgroup Metabolite abbreviation Metabolite name N analyzed in the study cohorts VLDL XXL.VLDL.PL Phospholipids in chylomicrons and extremely large VLDL 26,065 XXL.VLDL.L Total lipids in chylomicrons and extremely large VLDL 26,065 XXL.VLDL.P Concentration of chylomicrons and extremely large VLDL particles 26,065 XXL.VLDL.TG Triglycerides in chylomicrons and extremely large VLDL 26,065 XL.VLDL.PL Phospholipids in very large VLDL 26,065 XL.VLDL.TG Triglycerides in very large VLDL 26,065 XL.VLDL.L Total lipids in very large VLDL 26,065 XL.VLDL.P Concentration of very large VLDL particles 26,065 L.VLDL.C Total cholesterol in large VLDL 26,065 L.VLDL.FC Free cholesterol in large VLDL 26,065 L.VLDL.PL Phospholipids in large VLDL 26,065 L.VLDL.TG Triglycerides in large VLDL 26,065 L.VLDL.CE Cholesterol esters in large VLDL 26,065 L.VLDL.L Total lipids in large VLDL 26,065 L.VLDL.P Concentration of large VLDL particles 26,065 M.VLDL.C Total cholesterol in medium VLDL 26,065 M.VLDL.FC Free cholesterol in medium VLDL 26,065 M.VLDL.PL Phospholipids in medium VLDL 26,065 M.VLDL.TG Triglycerides in medium VLDL 26,065 M.VLDL.CE Cholesterol esters in medium VLDL 26,065 M.VLDL.L Total lipids in medium VLDL 26,065 M.VLDL.P Concentration of medium VLDL particles 26,065 S.VLDL.C Total cholesterol in small VLDL 26,065 S.VLDL.FC Free cholesterol in small VLDL 26,065 S.VLDL.PL Phospholipids in small VLDL 26,065 S.VLDL.TG Triglycerides in small VLDL 26,065 S.VLDL.L Total lipids in small VLDL 26,065 S.VLDL.P Concentration of small VLDL particles 26,065 XS.VLDL.PL Phospholipids in very small VLDL 26,065 XS.VLDL.TG Triglycerides in very small VLDL 26,065 XS.VLDL.L Total lipids in very small VLDL 26,065 XS.VLDL.P Concentration of very small VLDL particles 26,065 IDL IDL.FC Free cholesterol in IDL 26,065 IDL.PL Phospholipids in IDL 26,065 IDL.L Total lipids in IDL 26,065 IDL.P Concentration of IDL particles 26,065 IDL.TG Triglycerides in IDL 26,065 IDL.C Total cholesterol in IDL 26,065 LDL L.LDL.C Total cholesterol in large LDL 26,065 L.LDL.FC Free cholesterol in large LDL 26,065 L.LDL.PL Phospholipids in large LDL 26,065 L.LDL.CE Cholesterol esters in large LDL 26,065 L.LDL.L Total lipids in large LDL 26,065 L.LDL.P Concentration of large LDL particles 26,065

17 Lipids Fatty acids M.LDL.C Total cholesterol in medium LDL 26,065 M.LDL.PL Phospholipids in medium LDL 26,065 M.LDL.CE Cholesterol esters in medium LDL 26,065 M.LDL.L Total lipids in medium LDL 26,065 M.LDL.P Concentration of medium LDL particles 26,065 S.LDL.C Total cholesterol in small LDL 26,065 S.LDL.L Total lipids in small LDL 26,065 S.LDL.P Concentration of small LDL particles 26,065 HDL XL.HDL.C Total cholesterol in very large HDL 26,065 XL.HDL.FC Free cholesterol in very large HDL 26,065 XL.HDL.PL Phospholipids in very large HDL 26,065 XL.HDL.TG Triglycerides in very large HDL 26,065 XL.HDL.CE Cholesterol esters in very large HDL 26,065 XL.HDL.L Total lipids in very large HDL 26,065 XL.HDL.P Concentration of very large HDL particles 26,065 L.HDL.C Total cholesterol in large HDL 26,065 L.HDL.FC Free cholesterol in large HDL 26,065 L.HDL.PL Phospholipids in large HDL 26,065 L.HDL.CE Cholesterol esters in large HDL 26,065 L.HDL.L Total lipids in large HDL 26,065 L.HDL.P Concentration of large HDL particles 26,065 M.HDL.C Total cholesterol in medium HDL 26,065 M.HDL.FC Free cholesterol in medium HDL 26,065 M.HDL.PL Phospholipids in medium HDL 26,065 M.HDL.CE Cholesterol esters in medium HDL 26,065 M.HDL.L Total lipids in medium HDL 26,065 M.HDL.P Concentration of medium HDL particles 26,065 S.HDL.TG Triglycerides in small HDL 26,065 S.HDL.L Total lipids in small HDL 26,065 S.HDL.P Concentration of small HDL particles 26,065 VLDL.TG Triglycerides in VLDL 26,065 VLDL.D Mean diameter for VLDL particles 26,065 LDL.C Total cholesterol in LDL 26,065 LDL.D Mean diameter for LDL particles 26,065 HDL.C Total cholesterol in HDL 26,065 HDL2.C Total cholesterol in HDL2 (Lipido) 26,065 HDL3.C Total cholesterol in HDL3 (Lipido) 26,065 HDL.D Mean diameter for HDL particles 26,065 Serum.TG Serum total triglycerides 26,065 Serum.C Serum total cholesterol 26,065 EstC Esterified cholesterol 16,107 FreeC Free cholesterol 16,107 ApoA1 Apolipoprotein A-I (Lipido) 26,065 ApoB Apolipoprotein B (Lipido) 26,065 ApoB/ApoA1 Apolipoprotein B by apolipoprotein A-I (Lipido) 26,065 TG/PG Ratio of triglycerides to phosphoglycerides 16,107 TotPG Total phosphoglycerides 16,107 FAw3 Omega-3 fatty acids 16,107 FAw6 Omega-6 fatty acids 16,107

18 Amino acids Small molecules, energy metabolism FAw79S Omega-7, omega-9 and saturated fatty acids 16,107 FAw3/FA Ratio of omega-3 fatty acids to total fatty acids 16,107 FAw6/FA Ratio of omega-6 fatty acids to total fatty acids 16,107 FAw3/FAw6 Ratio of omega-3 to omega-6 fatty acids 16,107 Ratio of omega-7, omega-9 and saturated fatty acids to total fatty FAw79S/FA acids 16,107 LA 18:2, linoleic acid 16,107 PUFA Other polyunsaturated fatty acids than 18:2 16,107 DHA 22:6, docosahexaenoic acid 16,107 MUFA Monounsaturated fatty acids; 16:1, 18:1 16,107 PC Phosphatidylcholine and other cholines 16,107 SM Sphingomyelins 16,107 TotFA Total fatty acids 16,107 Description of average fatty acid chain length, not actual carbon FALen number 16,107 CH2 in FA Average number of methylene groups in a fatty acid chain 16,107 CH2 in DB Average number of methylene groups per a double bond 16,107 DB in FA Average number of double bonds in a fatty acid chain 16,107 BIS in DB Ratio of bisallylic groups to double bonds 16,107 BIS in FA Ratio of bisallylic groups to total fatty acids 16,107 MobCH Double bond protons of mobile lipids 26,065 MobCH2 CH2 groups of mobile lipids 26,065 MobCH3 CH3 groups of mobile lipids 26,065 Ala Alanine 26,065 Gln Glutamine 16,107 Gly Glycine 26,065 His Histidine Ile Isoleucine 26,065 Leu Leucine 26,065 Phe Phenylalanine 26,065 Tyr Tyrosine 26,065 Val Valine 26,065 Cit Citrate 26,065 Crea Creatinine 26,065 Glc Glucose 26,065 Alb Albumin 26,065 Pyr Pyruvate 26,065 Gp Glycoprotein acetyls, mainly a1-acid glycoprotein 26,065 bohbut 3-hydroxybutyrate 26,065 Ace Acetate 26,065 AcAce Acetoacetate 26,065 Lac Lactate 26,065 Glol Glycerol 16,107 Urea Urea

19 Supplementary Table 2. Age-sex-interaction in random effects meta-analysis of three Finnish cohorts among year-old men and women (n=3,478).analysis utilized multivariate linear regression. Metabolite β (CI 95%) P-value Lipoproteins and lipids Concentration of chylomicrons and largest VLDL particles ( ) Concentration of very large VLDL particles ( ) Concentration of large VLDL particles ( ) Concentration of medium VLDL particles ( ) Concentration of small VLDL particles ( ) Concentration of very small VLDL particles ( ) 8.08E-07 Concentration of IDL particles ( ) 8.34E-07 Concentration of large LDL particles ( ) Concentration of medium LDL particles ( ) Concentration of small LDL particles ( ) Concentration of very large HDL particles ( ) Concentration of large HDL particles ( ) Concentration of medium HDL particles ( ) Concentration of small HDL particles ( ) Triglycerides in VLDL ( ) Mean diameter of VLDL particles ( ) Total cholesterol in LDL ( ) Mean diameter of LDL particles ( ) Total cholesterol in HDL ( ) Total cholesterol in HDL (8.00E ) Total cholesterol in HDL ( ) Mean diameter of HDL particles ( ) Serum total triglycerides ( ) Serum total cholesterol ( ) Esterified cholesterol ( ) 2.29E-08 Free cholesterol ( ) Apolipoprotein A ( ) Apolipoprotein-B ( ) Apolipoprotein-B/apolipoprotein A-1 ratio ( ) 0.774

20 Fatty acids and related measures Amino acids Total phophoglycerides ( ) Triglycerides/phophoglycerides ratio ( ) Omega-3 fatty acids ( ) Omega-6 fatty acids (9.00E ) Omega-7 and -9 and saturated fatty acids ( ) Omega-3 fatty acids/total fatty acids ratio ( ) Omega-6 fatty acids/total fatty acids ratio ( ) Omega-7 and -9 and saturated fatty acids/total fatty acids ratio ( ) Omega-3 fatty acids/omega-6 fatty acids ratio ( ) Linoleic acid (18:2) ( ) Docosahexanoid acid (22:6) ( ) Polyunsaturated fatty acids (other than 18:2) ( ) Monounsaturated fatty acids ( ) Total fatty acids ( ) Average fatty acid chain length ( ) Average number of CH2 groups in a fatty acid chain ( ) Average number of CH2 groups per double bond ( ) Average number of double bonds in a fatty acid chain ( ) Bisallylic groups/boudle bonds ratio ( ) Bisallylic groups/total fatty acids ratio ( ) Double bond protons of mobile lipids ( ) CH2 groups of mobile lipids ( ) CH3 groups of mobile lipids ( ) Phophatidyl choline and other cholines ( ) Sphingomyelins ( ) Alanine ( ) Glutamine ( ) 6.35E-06 Glycine ( ) Histidine ( ) 8.41E-06 Isoleucine (-1.00E ) Leucine ( ) Phenylalanine ( ) 0.697

21 Small molecules Tyrosine ( ) Valine ( ) Acetate ( ) Acetoacetate ( ) Citrate ( ) Creatinine ( ) Glucose ( ) Glycerol ( ) Glycoproteins acetyls (mainly a1-acid glycoprotein) ( ) Lactate ( ) Pyryvate ( ) Urea ( ) Albumin ( ) hydroxybutyrate ( ) The values are for age*sex-interaction term in a linear regression model adjusted for BMI, daily smoking, and fasting time before sampling.

22 Supplementary Table 3. Association of menopause status with the metabolites in random effects meta-analysis using multivariate linear regression. Women of all age groups (n=10,083) year-old women (n=3,204) Metabolite β (CI 95%) P-value β (CI 95%) P-value Lipoproteins and lipids Concentration of chylomicrons and largest VLDL particles 0.16 ( ) ( ) Concentration of very large VLDL particles 0.29 ( ) ( ) Concentration of large VLDL particles 0.24 ( ) ( ) Concentration of medium VLDL particles 0.18 ( ) ( ) Concentration of small VLDL particles 0.19 ( ) ( ) Concentration of very small VLDL particles 0.21 ( ) ( ) Concentration of IDL particles 0.25 ( ) ( ) Concentration of large LDL particles 0.24 ( ) ( ) Concentration of medium LDL particles 0.21 ( ) ( ) Concentration of small LDL particles 0.20 ( ) ( ) Concentration of very large HDL particles ( ) ( ) Concentration of large HDL particles 0.17 ( ) ( ) Concentration of medium HDL particles 0.14 ( ) ( ) Concentration of small HDL particles 0.21 ( ) ( ) Triglycerides in VLDL 0.19 ( ) (-7.00E ) Mean diameter of VLDL particles 0.13 ( ) ( ) Total cholesterol in LDL 0.22 ( ) ( ) Mean diameter of LDL particles ( ) ( ) Total cholesterol in HDL 0.24 ( ) ( ) Total cholesterol in HDL ( ) ( ) Total cholesterol in HDL ( ) ( ) Mean diameter of HDL particles ( ) ( ) Serum total triglycerides 0.20 ( ) ( ) Serum total cholesterol 0.36 ( ) ( ) Esterified cholesterol 0.42 ( ) ( ) Free cholesterol 0.39 ( ) 2.72E ( ) Apolipoprotein A ( ) ( ) 0.303

23 Fatty acids and related measures Amino acids Apolipoprotein-B 0.26 ( ) 7.49E ( ) Apolipoprotein-B/apolipoprotein A-1 ratio ( ) ( ) Total phophoglycerides 0.47 ( ) ( ) Triglycerides/phophoglycerides ratio 0.27 ( ) 2.43E ( ) Omega-3 fatty acids 0.45 ( ) ( ) Omega-6 fatty acids 0.60 ( ) ( ) Omega-7 and -9 and saturated fatty acids 0.54 ( ) ( ) Omega-3 fatty acids/total fatty acids ratio 0.11 ( ) ( ) Omega-6 fatty acids/total fatty acids ratio ( ) ( ) Omega-7 and -9 and saturated fatty acids/total fatty acids ratio 0.16 ( ) ( ) Omega-3 fatty acids/omega-6 fatty acids ratio 0.18 ( ) ( ) Linoleic acid (18:2) 0.39 ( ) ( ) Docosahexanoid acid (22:6) 0.43 ( ) ( ) Polyunsaturated fatty acids (other than 18:2) 0.51 ( ) ( ) Monounsaturated fatty acids 0.43 ( ) ( ) Total fatty acids 0.58 ( ) ( ) Average fatty acid chain length ( ) ( ) Average number of CH2 groups in a fatty acid chain 0.22 ( ) ( ) Average number of CH2 groups per double bond 0.32 ( ) ( ) Average number of double bonds in a fatty acid chain ( ) ( ) Bisallylic groups/boudle bonds ratio ( ) ( ) Bisallylic groups/total fatty acids ratio ( ) ( ) Double bond protons of mobile lipids 0.45 ( ) ( ) CH2 groups of mobile lipids 0.40 ( ) ( ) CH3 groups of mobile lipids 0.42 ( ) ( ) Phosphatidyl choline and other cholines 0.39 ( ) ( ) Sphingomyelins 0.33 ( ) 1.87E ( ) Alanine 0.24 ( ) ( ) Glutamine 0.24 ( ) 1.27E ( ) Glycine ( ) ( ) Histidine 0.20 ( ) ( ) Isoleucine 0.21 ( ) ( )

24 Small molecules Leucine 0.22 ( ) ( ) Phenylalanine 0.41 ( ) ( ) Tyrosine 0.19 ( ) 4.76E ( ) Valine ( ) ( ) Acetate ( ) ( ) Acetoacetate ( ) ( ) Citrate ( ) ( ) Creatinine ( ) ( ) Glucose ( ) ( ) Glycerol ( ) ( ) Glycoprotein acetyls (mainly a1-acid glycoprotein) 0.48 ( ) ( ) Lactate ( ) ( ) Pyryvate ( ) ( ) Urea ( ) ( ) Albumin 0.13 ( ) ( ) hydroxybutyrate ( ) ( ) The values are for menopause status in a linear regression model adjusted for age, BMI, daily smoking, and fasting time before sampling.

25 Supplementary Table 4. Comparison of metabolite levels among Finnish year-old hormone replacement therapy users (n=813) and postmenopausal non-users (n=735). Metabolite β (CI 95%) P-value Apolipoprotein-B/Apolipoprotein A1 -ratio 0.28 ( ) 1.22E-08 Concentration of IDL particles 0.27 ( ) 2.68E-08 Albumin 0.25 ( ) 9.04E-07 Concentration of very small VLDL particles 0.21 ( ) 4.65E-06 Concentration of large HDL particles ( ) 1.57E-05 Concentration of very large VLDL particles 0.19 ( ) 3.12E-05 Apolipoprotein-B 0.26 ( ) 4.24E-05 Glycoprotein acetyls 0.17 ( ) Total cholesterol in HDL ( ) Total cholesterol in HDL ( ) Mean diameter of HDL particles ( ) Concentration of large LDL particles 0.27 ( ) Figures are from random effects meta-analysis performed with multivariate linear regression model, adjusted for BMI, daily smoking status, age, and fasting time.

26 Supplementary Table 5. Age effects on metabolites among year-old Finnish women (n=1,804). Metabolite β (95% CI) P-value CH3-groups in mobile lipids ( ) 7.45e-24 Total phosphiglycerides ( ) 1.53e-22 Apolipoprotein-B ( ) 8.67e-20 Sphingomyelins ( ) 1.66e-19 Total fatty acids ( ) 4.43e-19 Apolipoprotein A ( ) 1.53e-16 CH2-groups in mobile lipids ( ) 1.40e-16 Omega-6 fatty acids ( e-15 Monounsaturated fatty acids (16:1, 18:1) ( ) 2.52e-15 Linoleic acid (18:2) ( ) 4.21e-13 Serum triglycerides ( ) 4.49e-13 Concentration of small VLDL particles ( ) 2.58e-11 Free cholesterol ( ) 1.83e-10 Concentration of very large VLDL particles ( ) 5.59e-09 Esterified cholesterol ( ) 1.93e-08 Polyunsaturated fatty acids (other than 18:2) ( ) 3.84e-08 Omega-7 and -9 and saturated fatty acids ( ) 5.34e-08 Concentration of large VLDL particles ( ) 9.23e-08 Phenylalanine ( ) 1.08e-07 Phosphatidyl choline and other cholines ( ) 1.24e-07 Total cholesterol in HDL ( ) 1.33e-07 Serum total cholesterol ( ) 2.76e-07 Concentration of very small VLDL particles ( ) 2.78e-07 Glycoprotein acetyls, mainly a-1 acid glycoprotein ( ) 6.36e-07 Double bond protons of mobile lipids ( ) 8.06e-07 Concentration of small LDL particles ( ) 8.20e-07 Glucose ( ) 1.45e-06 Alanine ( ) 7.02e-06 Concentration of medium LDL particles ( ) 7.75e-06 DHA ( ) 8.80e-06 Glutamine ( ) 8.82e-06

27 Total cholesterol in HDL ( ) 9.34e-06 Concentration of IDL particles ( ) 9.46e-06 Concentration of large LDL particles ( ) 3.31e-05 Concentration of medium VLDL particles ( ) 4.01e-05 Apolipoprotein-B/Apolipoprotein A-1 -ratio ( ) 4.07e-05 Omega-3 fatty acids ( ) 4.26e-05 Average fatty acid chain length ( ) 6.56e-05 Tyrosine ( ) Concentration of large HDL particles ( ) Mean diameter of HDL particles ( ) Urea ( ) Glycerol ( ) Leucine ( ) Figures are for one year increase in age from random effects meta-analysis performed with multivariate linear regression adjusted for daily smoking, BMI, and fasting time.

28 Supplementary Table 6. Fasting effects on metabolites among year-old Finnish women (n=1,490) Metabolite β (95% CI) P-value Tyrosine ( ) 1.57e-07 CH2-gourps per double bond ( ) Valine ( ) Free cholesterol ( ) Figures are for one hour shift in fasting time from random effects meta-analysis adjusted for age, daily smoking, BMI. Multivariate linear regression model was utilized.

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