Claus Cornett. Biofluids

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1 Biofluids

2 General remarks Some Characteristics of Biofluids Water sig. Viscosity Protein Lipid Overlap Rel. Ionic Strengt h ph - range Urine Bile Blood plasma Whole Blood CSF Milk Saliva Gastric Juice >1-2 Pancreatic Juice Seminal fluid (>7.0) 7.7 Prostatic fluid Seminal vesicle fluid Amniotic fluid Like serum Follicular fluid Synovial Fluid Aqueous Humor After Nicholson et al Additional inf from Principles of Mammalian Biochemistry, 7th ed Lipowitz, Biofluids

3 General remarks Some Characteristics of Biofluids Water sig. Viscosity Protein Lipid Overlap Rel. Ionic Strength ph - range Urine Bile Blood plasma Whole Blood CSF Milk Saliva Gastric Juice < 1 Pancreatic Juice Seminal fluid Prostatic fluid Seminal vesicle fluid Amniotic fluid Like serum Follicular fluid Synovial Fluid Aqueous Humor

4 General remarks Water Signal BIG signal Water Supression 4

5 General remarks Some Characteristics of Biofluids Water sig. Viscosity Protein Lipid Overlap Rel. Ionic Strengt h ph - range Urine Bile Blood plasma Whole Blood CSF Milk Saliva Gastric Juice < 1 Pancreatic Juice Seminal fluid Prostatic fluid Seminal vesicle fluid Amniotic fluid Like serum Follicular fluid Synovial Fluid Aqueous Humor

6 General remarks Viscosity Linewidth Diffusion Sharp peaks Better resolution and better S/N Signal/Noise W FWHH = 1 π T 2 τ c = 0.16 η k T V (Spherical molecules). Ando&Webb 6

7 General remarks Viscosity Linewidth Diffusion Sharp peaks Better resolution and better S/N Signal/Noise 1 W = FWHH π T 2 τ c = 0.16 η k T V (Spherical molecules). Ando&Webb We have pulse sequences, that can distinguish based on T 2 7

8 General remarks Viscosity Linewidth Diffusion Signal/Noise D = k T Size of molecule 6 π η r We have pulse sequences, that can distinguish based on T 2 8

9 General remarks Some Characteristics of Biofluids Water sig. Viscosity Protein Lipid Overlap Rel. Ionic Strength ph - range Urine Bile Blood plasma Whole Blood CSF Milk Saliva Gastric Juice < 1 Pancreatic Juice Seminal fluid Prostatic fluid Seminal vesicle fluid Amniotic fluid Like serum Follicular fluid Synovial Fluid Aqueous Humor

10 General remarks Protein Overlap Visibility protein binding (reversible/irreversible) 10

11 General remarks Some Characteristics of Biofluids Water sig. Viscosity Protein Lipid Overlap Rel. Ionic Strength ph - range Urine Bile Blood plasma Whole Blood CSF Milk Saliva Gastric Juice < 1 Pancreatic Juice Seminal fluid Prostatic fluid Seminal vesicle fluid Amniotic fluid Like serum Follicular fluid Synovial Fluid Aqueous Humor

12 General remarks Lipid Overlap Viscosity Multiphase systems (Vesicles, Liquid Crystals etc.) Bad lineshapes (homogeneity, differences in magnetic susceptibility) 12

13 General remarks Some Characteristics of Biofluids Water sig. Viscosity Protein Lipid Overlap Rel. Ionic Strength ph - range Urine Bile Blood plasma Whole Blood CSF Milk Saliva Gastric Juice < 1 Pancreatic Juice Seminal fluid Prostatic fluid Seminal vesicle fluid Amniotic fluid Like serum Follicular fluid Synovial Fluid Aqueous Humor

14 General remarks Overlap 2D: Coupling constant 1D: Physicochemical properties/spectral Editing 14

15 Copyright Biofluids General remarks Overlap 2D: Coupling constant Do not depend on B 0 J-resolved (Separate couplings and Chemical shift) OBS some suppression of large molecules COSY-type Spectra Coupling network (identification) HETCOR Heteronuclear correlation isotope substitution, -other -omes 15

16 General remarks Overlap 1D: Physicochemical properties/spectral Editing Do not depend on B 0 CPMG T 2 -Relaxtion linewidth (Diffusion) Small/Fast molecules Diffusion edited Large/Slow molecules HR-MAS with chromatographic material in rotor LogP etc. 16

17 General remarks Some Characteristics of Biofluids Water sig. Viscosity Protein Lipid Overlap Rel. Ionic Strength ph - range Urine Bile Blood plasma Whole Blood CSF Milk Saliva Gastric Juice < 1 Pancreatic Juice Seminal fluid Prostatic fluid Seminal vesicle fluid Amniotic fluid Like serum Follicular fluid Synovial Fluid Aqueous Humor

18 18 Biofluids General remarks ph [rel] tautit ph = d: taurin + O O tautit d: taurin H3 N H 2 N S O O - S ph = O O - tautit d: taurin ph = 9.99 tautit d: taurin ph = ºC, pk = 9.04 tautit d: taurin (37ºC, pk = 8.6) ph = 9.23 tautit d: taurin Scale : 2.48 ph = 8.91 tautit d: taurin Scale : 2.04 ph = 8.73 tautit d: taurin Scale : 1.89 ph = 8.53 tautit d: taurin Scale : 2.06 ph = 8.27 tautit d: taurin Scale : 2.16 ph = 7.86 tautit d: taurin Scale : 2.18 ph = 7.63 tautit d: taurin Scale : 2.27 ph = 3.12 tautit d: taurin Scale : 2.38 ph = 2.18 tautit d: taurin Scale : 2.63 ph = 1.79 Copyright

19 Blood Plasma 19 Water Viscosity UrineProtein Lipid Overlap Rel. sig. Ionic Strengt h ph - range Urine Bile Blood plasma Whole Blood CSF Milk Saliva Water sig. Viscosity Protein Lipid Overlap Rel. Ionic Strength Gastric Juice < 1 Pancreatic Juice ++ Blood ++ plasma Seminal fluid Prostatic fluid Seminal vesicle fluid Amniotic fluid Like serum Follicular fluid Synovial Fluid Aqueous Humor Water sig. Viscosity Protein Lipid Overlap Rel. Ionic Strength

20 Blood Plasma Define Blood... Whole Blood vs Blood Plasma vs. Blood Serum Snapshot 20

21 Blood Plasma Define Blood... Whole Blood several different cell typer: Erythrocytes (Red blood cells) Leukocytes (White blood cells) Thrombocytes (platelets) 21

22 Blood Plasma Define Blood... Hematocrit ph po2 pco 2 HCO - 3 Oxygen saturation 45 ± 7 (38-52%) for males 42 ± 5 (37-47%) for females 10-13kPa ( mmhg) 4.8kPa - 5.8kPa (35-45 mmhg) 21mM - 27mM Oxygenated: 98-99% Deoxygenated: 75% 22

23 Blood Plasma Define Blood... 23

24 Blood Plasma Define Blood... Hematocrit 45 ± 7 (38-52%) for males 42 ± 5 (37-47%) for females Ht, HCT, Packed Cell Volume (PCV) 24

25 Blood Plasma Define Blood... Plasma About 55% of whole blood is Blood Plasma. Straw-yellow in color litres in an average human 92% water 8% blood plasma proteins (Serum Albumin, Clotting factors, Immunoglobulins, Lipoproteins a.o.) And a wee bit of other stuff: Glucose, Amino Acids, Fatty Acids, CO 2, Lactic Acid, Electrolytes (See. Human Metabolome Database: Biofluid_browse.cgi?browsn=1&sortby=5 ) 25

26 Blood Plasma Define Blood... Serum Plasma from which the clotting proteins have been removed. Most of the proteins remaining are albumin and immunoglobulins 26

27 Blood Plasma Chylomicrons carry triacylglycerol (fat) from the intestines to the liver, skeletal muscle, and to adipose tissue (diameter 75 to 1,200 nm). Very low density lipoproteins (VLDL) - carry (newly synthesised) triacylglycerol from the liver to adipose tissue. Intermediate density lipoproteins (IDL) - are intermediate between VLDL and LDL. They are not usually detectable in the blood. Low density lipoproteins (LDL) - carry cholesterol from the liver to cells of the body. Sometimes referred to as the "bad cholesterol" lipoprotein. High density lipoproteins (HDL) - collects cholesterol from the body's tissues, and brings it back to the liver. Sometimes referred to as the "good cholesterol" lipoprotein. These are further subdivided... Changes are characteristic for diseased states... 27

28 Blood Plasma Combined hyperlipidemia (or -aemia). Commonly occurring form of hypercholesterolemia. Increased LDL and triglyceride concentrations, often accompanied by decreased HDL. 28

29 Blood Plasma - Sample Prep Problems. Broad plasma lipoprotein/ fat profiles Severe overlap Not many small molecules visible Still a lot of water... 29

30 SaBiofluids Blood Plasma - Sample Prep Solutions. Standard sample preparation: Add one part 0.9 % saline (10-100%) D 2 O to two parts plasma/serum. If for NMR tubes centrifuge 5 min, 12,000g, 4ºC Run just with presaturation (e.g. Noesypresat 1D) Fade out the Big molecules (CPMG 1D). Sort by T 2, also reduces S/N of small molecules. Fade out small molecules (Diffusion edited 1D). Sort by molecular mobility, slow molecules remains also reduces signals from slow moving molecules. HR MAS 30

31 Blood Plasma - Sample Prep AND/OR Precipitate proteins e.g. by means of acetonitrile or perchloric acid (beware of ionic strength!). Supernatant can be run either directly or after freezedrying+reconstitution (allows for upconcentration, lose volatiles). Only presat spectra relevant 31

32 Blood Plasma Diffusion edited CPMG (T 2 ) 1D noesypresat (app. 8 min) ppm 32

33 Blood Plasma Precipitated with MeCN:plasma 2:1. Centrifuged, supernatant freezedried, reconstituted with 600 ul 0.33 M phosphate buffer ph 7.4. (0.5 ml plasma). (residual MeCN at δ 2.09 ppm. Expt ca. ½ hour MHz ppm ppm

34 Cerebrospinal Fluid (CSF) 34 Water Viscosity UrineProtein Lipid Overlap Rel. sig. Ionic Strengt 4 h ph - range Urine Bile Blood plasma Whole Blood CSF Milk Saliva Water sig. Viscosity Protein Lipid Overlap Rel. Ionic Strength Gastric Juice < 1 Pancreatic Juice CSF Seminal fluid Prostatic fluid Seminal vesicle 3 fluid Amniotic fluid Like serum Follicular fluid Synovial Fluid Aqueous Humor Water sig. Viscosity Protein Lipid Overlap Rel. Ionic Strength

35 Cerebrospinal Fluid (CSF) Cerebospinal fluid (CSF) Plasma filtrate + secretion (Choroid Plexus) - Plasma lipoproteins Glucose generally lower Amino Acids Always lower 35

36 Cerebrospinal Fluid (CSF) 36

37 Cerebrospinal Fluid (CSF) Composition similar to blood plasma (CSF is filtered plasma) with some differences: Higher levels of specific electrolytes, such as magnesium, sodium, chloride, and hydrogen. Levels of other electrolytes such as calcium and potassium. Amino Acids, Carbohydrates etc. (See e.g. Humane Metabolome Database, Biofluid_browse.cgi?browsn=5&sortby=5) Sample Storage (Levine et. al. 2000). : Below 20º C if deproteinized and neutralized Othwerwise below- 80º C. 37

38 Cerebrospinal Fluid (CSF) Dunne et al ph: 7.6 ± 0.2 O 2 : 140 ± 40 mmhg CO 2 : 28 ± 12 mmhg Lactate: 1.5 ± 1.0 mm Glucose: 3.7 ± mm 38

39 Cerebrospinal Fluid (CSF) Sample preparation. Early works: Freeze drying, reconstitution with same volume of D 2 O with TSP. Later works No freeze drying, D 2 O with TSP added. Sample preparation (Levine et. al. 2000). Samples were concentrated by lyophilization and reconstitued in 99.8 % D 2 O With TSP. ph Justeret til 5.1 Sample Preparation (Lutz et. al, 1998). Lyophilized samples from several patients were pooled, concentration factor 10. Levine, J., Et al. J. Neural Transmission. 107, (2000), Lutz, N.W., Eta Al. FEBS Letters, 425, (1998),

40 Cerebrospinal Fluid (CSF) Engelke, U.F.H., Et al. NMR in Biomedicine. 18, (2005),

41 Cerebrospinal Fluid (CSF) 1. TSP 2. 2.Leucine, Isoleucine, Valine 3. Isobutyrate 4. β-hydroxybutyrate 5. Lactate 6. Alanine 7. Acetate 8. Valine 9. Glutamine (small amount of Glutamate) 10. Succinate 11. Citrate 12. Dimethylamine 13. From plastic? 14. Glucose+small amount of Myoinositol, α-amino acids 15. β-glucose 16. Tyrosine 17. Phenylalanine 18. Histidine 19. Adenosine 20. Formiate NS = 16, LB = 0.5 Hz SF 600 MHz, TE = 298 K Dunne, V.G. et. al. NMR in Biomoedicine, 18, (2005),

42 Cerebrospinal Fluid (CSF) Dunne, V.G. et. al. NMR in Biomoedicine, 18, (2005),

43 Cerebrospinal Fluid (CSF) 43

44 Amniotic Fluid Water Viscosity UrineProtein Lipid Overlap Rel. sig. Ionic Strength ph - range 4 Urine Bile Blood plasma Whole Blood CSF Milk Saliva Gastric Water Juice sig. Viscosity Protein +++ Lipid - Overlap ++ Rel. Ionic Strength ++++ < 1 Pancreatic Juice Seminal fluid Amniotic fluid Prostatic fluid Seminal vesicle fluid 3 Amniotic fluid Like serum Follicular fluid Synovial Fluid Aqueous Humor Water sig. Viscosity Protein Lipid Overlap Rel. Ionic Strength

45 Amniotic Fluid Characteristics: The amnion grows and begins to fill, mainly with water, around two weeks after fertilisation. After a further 10 weeks the liquid contains proteins, carbohydrates, lipids and phospholipids, urea and electrolytes, all which aid in the growth of the fetus. In the late stages of gestation much of the amniotic fluid consists of fetal urine. (wikipedia) 45

46 Amniotic Fluid Sample Storage (Graca, G. Et. Al. 2007): -20 C is acceptable (still large changes in Acetate) Below -70 C better. Sample Preparation (Graca, G. Et. Al. 2007). 500 MHz: 100 ml 0.5 M Sodium Phospahte buffer, ph 7.2 (D 2 O) to 500 ml sample. Centrifugation g, 25 C, 5 min. 550 ml to NMR tube. NMR: Noesypr1d. 800 Mhz: 1 ml sample Freeze dried, reconstituted to 720 ml D 2 O, 80 ml 0.24 % TSP in D 2 O. NMR: ZGPR. Other relevant eksperiments: CPMG, Diffusion edited. 46

47 Amniotic Fluid 90 -puls CPMG Diffusion 800 MHz 1H-NMR spectrum of Amniotic Fluid (Reconstituted). Graca, G. Et. Al. (2007). Graca, G. Et. Al. Analytical Chemistry. 79, (2007),

48 Synovial Fluid Water Viscosity Urine Protein Lipid Overlap Rel. sig. Ionic Strength ph - range 4 Urine Bile Blood plasma Whole Blood CSF Milk Saliva Gastric Water Juice sig. Viscosity Protein +++ Lipid - Overlap ++ Rel. Ionic ++++ Strength < 1 Pancreatic Juice Synovial Fluid Seminal fluid Prostatic fluid Seminal vesicle fluid 3 Amniotic fluid Follicular fluid Synovial Fluid Aqueous Humor Water sig. Viscosity Protein Lipid Overlap Rel. Ionic Strength

49 Synovial Fluid Plasma filtrate. Eletrolytes and readily diffusable substances exchange with plasma 49

50 Synovial Fluid Normal synovial fluid contains 3-4 mg/ml hyaluronan (hyaluronic acid), a polymer of disaccharides composed of D-glucuronic acid and D-N-acetylglucosamine joined by alternating β-1,4 and β-1,3 glycosidic bonds. Lubricin (glycoprotein) 50

51 Bile 51 Water Viscosity Urine Protein Lipid Overlap Rel. sig. Ionic Strength ph - range 4 Urine Bile Blood plasma Whole Blood CSF Milk Saliva Gastric Water Juice sig. Viscosity Protein +++ Lipid - Overlap ++ Rel. Ionic ++++ Strength < 1 Pancreatic Juice Bile - Bladder Seminal fluid Prostatic fluid Seminal vesicle fluid 3 Amniotic fluid Like serum Follicular fluid Synovial 1 Fluid Aqueous Humor Water sig. Viscosity Protein Lipid Overlap Rel. Ionic Strength

52 Bile The components of bile: Cholesterol Lecithin (a phospholipid) Bile pigments (bilirubin digluc-uronide) Bile salts (sodium glycocholate & sodium taurocholate) Bicarbonate ions Drug metabolites (and other metabolites) 52

53 Bile Cholesterol Deoxycholic Acid Taurocholic acid 53

54 General remarks Define BioFluid... And then there s extracts Perchloric Acid extracts (PCA!) Acetonitrile + Water Chloroform (Lipids) And Perfusates (organs and cell cultures) 54

55 General remarks Define BioFluid... Perfusate (Custodiol). After heart transplant. Perfusate (Custodiol). Before addition of heart Claus 7.0 Cornett ppm

Nuclear Magnetic Resonance (NMR) Spectroscopy

Nuclear Magnetic Resonance (NMR) Spectroscopy G. A. Nagana Gowda Northwest Metabolomics Research Center University of Washington, Seattle, WA 1D NMR Blood Based Metabolomics Histidine Histidine Formate