Isotopic analysis of metals like calcium, iron, zinc, copper, and sulfur can provide insights into human health and disease processes. Metal isotopes behave differently depending on their molecular bonding environments within the body and can be fractionated by metabolic pathways. Measurement of metal isotopes in tissues and biofluids using techniques like MC-ICP-MS has the potential to serve as biomarkers for conditions like osteoporosis, hemochromatosis, Wilson's disease, and cancers. For example, studies found heavier iron isotopes in patients with hemochromatosis and lighter copper isotopes in patients with Wilson's disease and some cancers.

1. MEDICAL APPLICATIONS OF ISOTOPES
METALLOMICS
Research Paper Author: Albarède, F. et.al (2017)
By
Sanjay Dubey
Guide: Dr.Ryoji Tanaka
Course : Volatile element geo-and Cosmo-chemistry
REARCH PAPER PRESENTATION
REF:Albarède, F., Télouk, P., & Balter, V. (2017). Medical Applications of Isotope Metallomics. Reviews in Mineralogy and Geochemistry, 82(1), 851-885.

2. Overview
• Introduction
• Basic feature of the paper
• The Isotope effect
• Key points
• Mass Spectrometer and MC-ICP-MS
• Use of Isotope study to Biological body
• An Overview of Ca, Fe, Zn Cu and S Biochemistry and Homeostasis
• Calcium Bone Loss
• Genetic an infectious Disease
• Isotope Metallomics in Cancer
• Assessing the potential of metal isotopes as Biomarkers
• Compartmentalizes models of Cellular Homeostasis

3. Introduction
Complexity of biology body and geological science
Biological complexity easy too understand compare to Earth science
Ref pp 6, An Introduction to Environmental Chemistry; Table 1, pp 116, Essentials of Medical Geology.
Different geological technique used in the application of Medical field.
Above data from ICP-MS
Molecular modeling properties like
configuration energy and isotopic properties
are restricted to small molecules

4. Basic feature of the paper
• Study of metal, metalloid or trace element of an
entire system and the interaction of the metal
with the organism’s other ‘omes’ such as the
genome, proteome or metabolome is Metallome
and subject is known as Metallomics
• Broad range of disciplines such as analytical,
bioinorganic, environmental or medicinal
chemistry involved.
• Paper discuss more about the application of
stable isotope in medicine
Fe, Cu, Zn, Ca and Mg play more biological specific role
E.g. Metalloprotein like Hemoglobin and Myoglobin
Only stable metal isotopic
which are closely related to
human body and other
organism studied in this
paper

5. The Isotopic Effect
Heisenberg Uncertainty Principle
PrincipleOrigin of Mass Dependent
Stable Isotopic Fractionation
Bond doesn’t come to rest even at
lowermost energy state. Isotopic
effect depend on temperature
Isotopic Effect is change in the small
energy induced by the isotopic
substitution
Three types of Isotopic effects
• Primary Isotope effect: bond to the
isotopic atom is broken
• Secondary isotope Effect: No direct
involvement of breaking/making bond
• Solvent Isotope Effect: Change in the
solving ability before and after
Isotope Fractionation: Variability in the
isotopic abundance in same element among
the co-existing species

6. Key points of isotopic effect
• Electron energy level do not alter due to the isotopic
substitution
• Properties that are dependent on the masses of the
bonded atoms get perturbed on isotopic substitution.
• For chemical purposes, the perturbation can be assumed
to be limited to vibrational frequencies
• vibrational energy associated with a bond is given by:
Eν = (v + ½) hν; v = 0, 1, 2, 3…… (v is the vibrational
quantum non-negative integer number)
• Favoring heavier isotope over lighter isotope in order to
minimize the total energy of the system
• Strength of the bond depend on the electronegativity and
ionization potential of opposite atom
• Bond with high Oxi.St and small C.N. prefer Heavy
isotopes.
• Process depend on the relative masses of isotopes E.g.
Effect is high for H isotopes due to twice increase in the
mass of element. Consequently, use of D and T has been
found to be invaluable in organic mechanistic research.
• Low activation energy tends to make lighter isotopes to
react faster than the heavier
Mass Spectrometer to
calculate Isotope abundance
(i) N in Amino acid
Histidine and O lactate
prefer heavy isotope (ii)
S bond in Cysteine
prefer light isotope.

7. Mass Spectrometer and MC-ICP-MS
• MS is an analytical technique that ionizes chemical species and sorts the ions based on their mass-to-charge ratio
• Variation of isotope abundance rarely exceeding one part per 1000 per unit mass difference.
Isotopic standard is taken from Vienna Standard Mean Ocean Water𝛿65 𝐶𝑢 = {
65
𝐶𝑢
63
𝐶𝑢
𝑠𝑎𝑚𝑝𝑙𝑒
65
𝐶𝑢
63
𝐶𝑢
𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑
- 1} *1000
• For Small Variation Measurement = High Transmission + Magnetic Mass filter
• Quadrupole Mass Spectrometer don’t posses high precision for the
measurement of natural abundance
• Mass fractionation in MS is done by replacing unknown sample by standard
one
• Heavy Isotope are kept in the numerator
• Difficult to ionize the metal such iron providing insufficient isotope
abundance data
• Double spike technique :Time Consuming and Limited applications
Working principle of MS
MC-ICP-MS Multiple collector inductively coupled plasma mass spectrometry
• High precision, Metal isotope abundance, Trace metal analyses (Yield Approx.
100% ) and Small sample (Nano-grams)

8. Use of Isotope study to Biological body
Why to study the isotope abundance instead of just measuring metal concentration in the body ?
A . Measurement of concentration is not acceptable for quantitative predication. Direction and magnitude of isotopic induced
bonding of metal with chelate can be predicted by using theory. E.g. Amino acid Cysteine or Histidine protein
B. Isotopes of given element can assist to predict the biochemical pathways due to their variation in the relative abundance
C. Density Functional Theory or DFT computational quantum mechanical model explaining the ground state orbital geometry
and vibrational frequency of metallic compound.
• Atom are confined in box and left to drift to attain stale molecular configuration
• Isotopes are substituted to infer the slight thermodynamic changes cause due to this
• Intensive calculation and consistent database
E.g. Cu in ceruloplasmin, a blood component and superoxide dismutase and etc.
D. Other theoretical method are used to overcome the limitation of DFT (i.e. Exchange and Correlation between electron).
Mossbauer spectroscopy can be used to measure nuclear kinetic energy .
E. Large proteins are so far beyond the reach of DFT but study have been made for fractionation of Cu, Fe, Zn and Ni in smaller
group of protein.
E.g. With the help of partition function the predicted δ65Cu value in Cu(II)(His)(H2O)42+ is 4.168 − 3.124 = 1.044 ‰ higher than
in Cu(II)(Cys)(H2O)42+.

9. Use of Isotope study to Biological body
Two enzymes use the two oxidation states
of copper to shuttle electrons. Copper is
bound to N from the amino acid histidine
(His, hard bond) and S from the amino acid
cysteine (Cys, soft bond).
Top transit : steady state
Bottom transit: Explaining different
pathways
Best Documented observed example:
• Cu and Zn isotopic fractionation
• Cu(II) heavy isotopes > Cu(I) light isotopes
• The e- (Strong electron negativity )
donor prefer heavy isotopes compare to
S(with weak e-vity )
• Zn with high low co-ordination number
prefer heavier isotopes
This type of study also can assist to
understand the isotope fractionation in the
large protein

10. Overview of Ca, Fe, Zn Cu and S Biochemistry and Hemostasis
• Strong electrostatic field and bare cation can be harmful to the many proteins. This is main reason to be attracted to metal
protein
• Topic is not completely based to understand the pathways of metal metabolism in Human Body
• Paper is to focus on the particular process (reaction, import and export, Oxid.state) to elucidate the mechanism of isotope
fractionation
CALCIUM
• Alkaline earth metal, Divalent and 6 Isotopes: 40, 42, 43, 44, 46,48. Ca in HB is 1.4 Kg and Daily intake is 1g
• 99% Body calcium stored in Bone in the form of Hydroxyapatite.
Important Points
• Calcium is particularly abundant in environment and variation of its isotopes were successfully investigated.
• Analysis by TIMS and MC-ICP-MS results:
• Ca becomes isotopically lighter as it moves through the food chain
• Bone contain light isotope of Ca. (Heavy Isotope of Ca isotope should favor high electronegativity group PO4
3- )
(Cartilage collagen prod. Chondrocytes Dead Chond. Replaced by Osteoblasts Bone building cell (O.B)
precipitate apatite ALP Enzyme Located in membrane of O.B. Liberated PO4
3- Apatite precipitation and
Mineralization i.e. Bone deposition .)
• Isotope fractionation reflect that Ca binding to the light ligand where Ca combine with phosphate, phosphate liberated
from phosphate ester which must avoid the precipitation of heavier Ca isotopes.

11. Overview of Fe, Zn Cu and S Biochemistry and Hemostasis
• Iron important to HB, Fe(II) responsible is the main carrier of O2 in the blood. HB contain 3g of Fe and Daily intake is 1-2mg.
• Iron present in the liver, kidney, spleen in the form of Fe(III) ferritin. A ferrihydrite wrapped in protein shell.
• Fe exist in HB in two Oxidation state Fe(II) and Fe(III)
• Iron 4 isotopes 54, 56, 57 and 58 abd Fe(III) bind both organic and inorganic ligand. Ferric hydroxide is highly insoluble.
• Iron isotope abundance are measured with MC-ICP-MS.
Iron homeostasis in Mammalian cells.
• Isotopic roles:
• Fe becoming Isotopically heavier after phlebotomy
• Fe quickly retrieve from ferritin to replace lost iron and Imp. Observation
in the treatment of anemia.
• Fe is isotopically heavier in the liver, spleen and bone morrow.
• Mechanism
• Fe(III) to Fe(II) by using DMT assisting Ferric to across the cell
membrance
• Fe(III) intestine transported by protein which bound to transferrin
iron carrier in the blood stream
• Intracellular iron stored in Ferritin
• Tf-Fe(III) receptor to Fe(II) cell digestible by using STEAP and DMT1

12. Overview of Zn Cu and S Biochemistry and Hemostasis
Zn
• Zn five isotopes : 64, 66, 67 and 68. Zn in HB is
1.5 to 3g & daily intake 10mg
• Organ: Cytosol and nucleus of the cell
• Excess Zn is bound to Metallothionein (Sulphur
rich protein)
• Function to maintain the balance of acid –base
cytosol and others like cell signaling pathways
and transcription factor.
Cu
• Cu has two isotopes 63 and 65 and two states Cu(I) and Cu(II)
• Cu in HB 50-150mg. Daily intake 1mg
• In all tissues and body fluids.
• Cu is stored in Metallothionien
S
• Sulfur in HB is 175g and Daily intake is 1g/d
• Sulphur in two amino acid cysteine and
methionine
• Important for the structure and stability of the
protein
• Main sulfur carrier is albumin
• Function : Hepran sulfate : Anticoagulant
substance commonly used as additive to lower
the viscosity of the blood and inhibit the blood
clotting.
Zn, Cu and S
homoeostasis
representation

13. Isotope composition of Fe-Zn-Cu-S in the blood cell of healthy person
• Cu and Zn is variable among the women and man.
• Cu is used biomarker to health status.
• Isotope composition of metals and sulfur in the organs and body fluids of mammal was unknown
• Isotope analysis done on mice, sheep and minipig
• Observation
• In mice Zn found isotopically heavy in blood and bone; light found in liver and brain.
• Cu light found in kidney
• Systematic analysis of Zn Cu and Fe isotopes in human body.
• Fe in RBC is isotopically light with respect to serum (protein). Whereas Zn and Cu are isotopically heavier ~
0.3% and 0.8% respectively.
• 𝛿66Zn and 𝛿65Cu difference was less than 0.2% in male and female of both serum and RBC.
• 𝛿65Cu is 0.2% heavier in men erythrocyte compare to woman.
• 66Zn/64Zn ration increase and 65Cu/63Cu ratio decrease with age.
• Fe isotope ratio in postmenopausal/anti hormonal conception women there characteristic are getting similar to
men.
• Isotopic pattern analysis show that residence time of metal into the body is Fe ~5.5 yrs. and Cu~20 days. For
both men and woman.
• 𝛿66Zn is higher around 0.15% for vegetarian relative to omnivorous and for Cu outcome was not so conclusive.
• 34S/32S done by EA-IRMS explain S in children serum is only slightly heavier but more scattered than S in adult Serum

14. Medical application of isotopes
Calcium and Bone loss
• Calcium isotopic variation in urine and blood sample. (To explain astronauts
suffer from bone loss)
• Ca increase in urine indicate lighter isotope which result due to metal liberated
by osteoclasts from the bones into blood serum.
• Loss of isotopically lighter Ca lead to bone mineralization response.
• Increase loss of Ca can also be result of enhanced expression of calcium-
binding protein. It can be the case of osteoporosis
• Calcium isotope analysis show the Ca fluxed in and out of bones without
adding isotope tracer to the diet.
Genetic and infectious disease
• Heavier Fe found to be in person suffering from hemochromatosis (Iron
overloading)
• Hemochromatosis contain 0.2-0.4% heavier iron than in healthy person
• Reason :Decrease in hepcidin protein lead to increase of iron in intestine
• Wilson disease is genetic disorder cause due to Cu metabolism lead to
physiological and neurological symptom
• Decrease in 𝛿65Cu and Cu in the serum.

15. Cu/Zn ratio : Application of Isotope Metallomics in Cancer
• Copper and Cu/Zn in the serum as indicators of
cancer status.
• The control group shows strong correlations,
reflecting the tight regulation of Zn concentrations in
the body (note that x/y = Zn).
• The trends for control men and women are different,
with men having, on average, less Cu and more Zn
than women..
• Copper remains stable in prostate cancer patients
relative to control men, but increases in colon cancer
patients.
• Zinc in the serum of breast cancer patients is
decreased and Cu probably increased relative to
healthy subjects.
• In breast cancer contain lighter Zn isotopic than the
blood, serum and healthy tissue.

16. Breast and colorectal cancer isotopic data analysis
• Evolution of serum δ65Cu in 140 samples taken
from 20 breast cancer cases until patient death
(Télouk et al. 2015).
• Each line represents a different patient, with
patterns used for differentiation purposes. The
shaded band (controls) represents the 75 % range
of δ65Cu in the serum of healthy donors
• Horizontal lines in the box: median; crosses: outliers.
• Separation between breast cancer patients and healthy
women is strong.
• Separation between breast cancer and colorectal cancer
patients and healthy men and women seems to depend
on mortality.

17. Medical application of isotopes
• Fig A Top: Isotopically heavy copper in tumor liver tissue relative to
normal tissue. The opposite direction of the changes in Cu isotope
abundances in serum and tumor may be explained in different ways.
• Fig A Bottom: Isotopically light copper and sulfur in the serum of
hepatocellular (liver cancer) carcinoma patients relative to controls
(Balter et al. 2015).
Fig A
• Biochemical Interpretation of
isotopic trends:
• Zn Isotope serum data show no
difference between cancer and
healthy donors of any age.
• in heavy 𝛿65Cu isotopic variation
in cancer patient is explained by
Cu chelated by lactate which
cancer cell produce massively
Extent of copper chelation by lactates
in the cytosol

18. Assessing the potential of metal isotopes as Bio-Markers
• Receiver Operating Characteristics (ROC) curve for δ65Cu in
the serum of cancer patients.
• The ROC curve plots the probability of a True Positive decision
vs the probability of False Positive decision. T
• The area under the ROC curve (AUC) may vary between 0.5
(pure chance) and 1.0 (fully trustworthy test)
• The value of 0.76 obtained for the present data set
supports the worth of δ65Cu as a diagnostic tool.
• The plot compares the δ65Cu values (left axis, black line) and the
molecular biomarkers (right axis):
• CEA (carcinoembryonic antigen, shade 1) and CA 15.3 (carbohydrate
antigens, shade
• The top bar scale shows the successive therapies received by the
patient.
• The copper isotope signal precedes the other markers by 2−3
months

19. Compartmentalized model of cellular Homeostasis
Mockup Example
• Zn transport through a cell. We assume that Zn is imported
and exported through transmembrane proteins.
• The number of these proteins may be modulated by cell
signaling. Transmembrane Zn transfer takes place by
diffusion.
• Excess Zn is stored in Zn-metallothionein, which requires
cysteine biosynthesis in the cytosol. Cysteine biosynthesis is
dependent on methionine uptake.
• Cytosolic cysteine is broken down with a first-order rate.
• All the units and the parameters are arbitrary.
• It was assumed that for time t the rate of metallothionein
catabolism increases, while the methionine concentration in
the extracellular medium decreases.
• Bottom panel: amounts of cytosolic Zn in each form. Top
panel: δ66Zn in the efflux.

20. Summary
• Cu has provided the strongest signal associated with a number of diseases, in particular cancer. Zinc, iron, and sulfur have not
so far proved to be as informative as copper.
• The exploratory stage of Cu isotope variations in blood has been very fruitful. Now that this field is becoming mature,
descriptive investigations need to be complemented.
• Among the upcoming challenges, several major questions need to be addressed, notably which part of the δ65Cu signal is due
to the cancer itself, and which part is due to other factors, such as age and, even more likely, inflammation.
• The isotopic study of copper will certainly add a new dimension to the understanding of chelation pathways and copper mass
balance, at the scale of both the cell and the organism, during the treatment.
• The prospects of isotope variations of magnesium, which is involved in a large number of biological segments, remain
unexplored.
• Molybdenum plays a role, notably through molybdenum hydroxylase, in a variety of hydroxylation, oxygen atom transfer, and
other oxidation–reduction reactions (Hille et al. 1998; Schwarz et al. 2009) and shows promise for future isotopic work. 879
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