Zinc estimation, plasma zinc . urine zinc, hair zinc, methods of estimation, Zinc dependent enzymes, compleximetric, fluorometric, anodic stripping method , Neutorn acativation analysis, Atomic absorption spectrometry , comparision of methods.

1. Laboratory Assessments
of Zinc
Hari Sharan Makaju
M.Sc. Clinical Biochemistry
1st year
2076/05/09

2. Laboratory Assessment of Zinc
Status
Although plasma zinc determination
Insensitive to dietary zinc intake
Subject to a variety of influences
the most widely used laboratory test
to confirm severe deficiency
to monitor adequacy of zinc provision,
Especially when interpreted together with changes in
serum albumin and the acute-phase response

3. Reliable biomarkers of zinc status
Plasma Zinc
Plasma samples are preferred to serum for
zinc analysis
Because of possible zinc contamination from
erythrocytes, platelets, and leukocytes during
clotting and centrifugation.
Measured by :
FAAS,
Spectrophotometric methods

4. Reliable biomarkers of zinc status
Zinc in Hair
Low hair zinc has been associated with poor
growth in children
Has been used as a criterion for initiating
supplementation studies.
Inconsistent results due to:
Variables such as Hair growth rate and external
contamination from hair dyes and cosmetics
Results from individual patients are difficult to
interpret.

5. Reliable biomarkers of zinc status
Urine Zinc
 A slight fall in the urine excretion of zinc has been
noted
 during dietary deficiency.
 Increased urine zinc is an important source of loss in
the severely injured catabolic patient
 Urine output increases with amino acid infusion given
during total parenteral nutrition

6. Others biomarkers of zinc status
Blood Cell Zinc
 Some investigators have suggested
 Zinc content of white cells and platelets better reflects
tissue zinc
 In experimental studies of zinc depletion in humans:
 Zinc content of neutrophils, lymphocytes, and platelets has
been shown to decline more rapidly than plasma zinc
 Required of large volume of blood and contamination
problems make difficulty especially in studies involving
children

7. Others biomarkers of zinc status
Blood Cell Zinc
Erythrocyte zinc has been suggested as an
alternative
Zinc intake during pregnancy
No changes in erythrocyte zinc were found
But Plasma zinc increased with intake.

8. Others biomarkers of zinc status
Zinc-Dependent Enzymes
 Despite the large number of zinc metalloenzymes that
have been identified,
 No single enzyme assay has yet found acceptance as an indicator
of zinc status.
 This may be result of :
 Avid retention of zinc by these enzymes, even in the face
of dietary zinc depletion,
 Difficulties with reproducible measurements of activity.
 Bone-specific alkaline phosphatase, extracellular SOD,
and lymphocyte and plasma 5-nucleotidase appear to
be responsive to zinc intake

9. Reference Intervals
 Serum zinc concentrations are generally 5 to 15%
higher than those of plasma
 Because of osmotic fluid shift from the blood
cells when various anticoagulants are used.
 Plasma zinc concentrations are decreased after food
and are higher in the morning than in the evening
 A reference interval for clinical guidance
 80 to 120 µg/dL (12 to 18 µmol/L)
 Urine zinc excretion
 0.2 to 1.3 mg/24 h (3 to 21 µmol/24 h)
 Fasting morning values of plasma zinc below 70 µg/dL
(10.7 µmol/L) on more than one occasion require
further investigation.
 Results below 30 µg/dL (5 µmol/L) suggest
likely defiiency.

10. Method for Zinc Estimation
Compleximetric method
Fluorometric method
Anodic stripping voltammetry
Atomic absorption spectrometry
Neutron activation analysis

11. Compleximetric method
Type of analysis
Colorimetric ,Quantitative
Principle :
After acid digestion (with mixture of high
purity nitric acid ,Perchloric and sulfuric acids
in ratio of 3:1:1 by volume) , zinc forms a
complex with dithizonate in carbon
tertrachloride , which is measured at 525nm.

12. Fluorometric method
Type of Analysis
Fluorometric , Quantitative
Principle :
The zinc – quinolinol complex are
stabilized by gum Arabic and
demonstrate a noticeable fluorescence at
517nm when excited at 375nm.

13. Anodic stripping voltammetry
 Type of Analysis
Electrochemical , Quantitative
Principle:
 The ionic zinc in serum is first reduced to elemental zinc by
negative potential applied to working electrode.
 The zinc is deposited onto this electrode as a mercury
amalgam.
 After a preselected time the working electrode potential is
changed to a more positive value, thereby causing the
oxidation of the zinc to the soluble Zn++
 The resulting anodic current produced by this oxidation is
measured and is proportional to the concentration of zinc

14. Atomic Absorption Spectrophotometry
 Type of analysis
Spectrophotometric , Quantitative
Principle:
 After deproteinization or dilution , specimen are
subjected to AAS determination
 Specimen are reacted at high temperature
,reducing the ionic zinc and converting it into
atomic zinc vapor and the light absorbed by the
zinc at its characteristic wavelength of 213.9nm is
quantified.
 Comment :
High specificity low minimum detection limit
Excellent precision and accuracy

15. Neutron activation analysis
 Type of analysis
 Radiochemical or nuclear
Principle
 68Zn is converted to unstable 69Zn by neutrons ; 69Zn decays
with release of gamma radiation at 0.439 MeV energy

16. Comparison of Assay
Dithizone Fluorometry Anodic
stripping
AAS-
Flame
AAA-
Tempr RT RT RT 2000oc 2000oc
PH >11 8.0 <1.0 <1.0 -
Linearity
(μg/l)
0 to 4000 0 to 4000 0 to 1000 0 to 2000 0 to 2000
Precision 5% 4% 5% 4% 3%
Time of
reaction
Hours Minutes Minutes Minutes Minutes
Specificity Low Low Adequate High High
Major
interference
Hg++,
Fe++,
Bi+++,
Cd++
Ca++, Mg++ Cu++ - -

17. References
 Tietz Textbook of CLINICAL CHEMISTRY MOLECULAR
DIAGNOSTICS, Carl A. Burtis, Ph.D., Edward R. Ashwood,
M.D,David E. Bruns, M.D, Fifth edition
 Methods in clinical chemistry , Kaplan L.A, Pesce A J
 Internet source.