Techniques of Analyses
i
TEC HNIQ UES O F ANALYSES IN
NUTRITIO NAL BIO C HEMISTRY
ii
iii
TEC HNIQ UES O F ANALYSES IN
NUTRITIO NAL BIO C HEMISTRY
Retno Murwani
Head of Labor at or y of Nut r it ional Biochemist r y
Dept . of Nut r it ion and Feed Science
Facul t y of Ani mal Science
Diponegor o Univer sit y Semar ang
Published by BP Undip
Semarang
November 2009
iv
Book : Techniques of Analyses in Nut rit ional Biochemist ry; Ret no
Murwani (aut hor); 1st Ed; Badan Penerbit Undi p Semarang; 2009
xii + 184 pages; 16. 5 x 23. 5 cm
ISBN : 978-979-704-838-9
Tit le:
TEC HNIQ UES O F ANALYSES IN
NUTRITIO NAL BIO C HEMISTRY
Ret no Murwani
copyright 2009
Al l r ight s r eser ved
No part of t his book may be reproduced in any f orm, by mimeograph
or any ot her means, wit hout permission in writ ing f rom t he aut hor
v
Preface
This book is writ t en t o document empirical experience during
t he course of various research grant s t hat t he aut hor has
received and carried out , as well as t o document experience
of more t han 23 years of t eaching as a lect urer. This book is
writ t en as part of World Class Universit y Program f or Book
Writ ing launched by Diponegoro Universit y in t he year 2009.
The aut hor is one of t welve universit y lect urers who are
assigned t o writ e t he book in English language (SK Rekt or
No. 520/ SK/ H7/ 2009).
This book is writ t en t o help st udent s and ot her readers in
carrying analyses eit her on t heir own, or as a ref erence and
guide in underst anding analyt ical met hods writ t en in scient if ic
j ournals t o support t heir research. The book consist s of t en
chapt ers, covering several maj or and minor nut rient s as well
as phyt onut rient such as polyphenol. It is beyond t he scope of
t his book t o cover all nut rient s known t oday. However, t he
principles and t echniques can be applied t o almost any
nut rient s. Each chapt er is int roduced wit h a paragraph t hat
gives a sample phrase which can ref lect t he need of a nut rient
analysis in t he cont ext of a scient if ic research t hat has been
published in scient if ic j ournals, or on a nut rient label of f eed
or f ood product s. This int roduct ion is meant t o ease t he
vi
reader t o relat e t o inf ormat ion or an inst ruct ion f ound in
many published works wit h t he mat erials given in each
chapt er. Af t er reading t his book t he readers wil l hopef ully be
able t o underst and writ t en analyt ical procedures and execut e
t he procedures int o pract ice in nut rit ion analyses.
It is my hope t hat t his book will be usef ul not only f or st udent s
in animal science f acult y, but also f or ot her st udent s and
readers who need a guide t o carry out nut rient analyses.
Grat ef ul acknowledgement is due t o t he f inancial support
f rom World Class Universit y Program f or
Book Writ ing,
Diponegoro Universit y, year 2009, and Prof . Dr. Ret mono, who
is a Prof essor in English Educat ion, as t he appoint ed expert f or
t he proof reading of t he English language.
Aut hor
Ret no Murwani
November, 2009
vii
Contents
Pref ace ........................................................................
v
Cont ent s ......................................................................
vii
Tables and Figures.......................................................
ix
I
Int roduct ion t o Nut rit ion Analysis................................
1
II
Unit s of A Nut rient Concent rat ion...............................
11
III
Principles of Spect rophot omet er .................................
31
IV
Det erminat ion of Lipid in Serum and Tissue ...............
61
V
Det erminat ion of Prot ein in Blood Serum ....................
87
VI
Det erminat ion of Phosphor in Feed or Food ................
95
VII
Det erminat ion of Calcium ...........................................
115
VIII
Det erminat ion of DNA and RNA From Cells or Tissue
123
of an Animal ...............................................................
IX
Det erminat ion of Enzyme Act ivit y...............................
133
X
Det erminat ion of Tot al Phenolic Compound ................
145
Ref erences ...................................................................
157
Appendix A
At omic Weight s of The Element s ………………………………
viii
163
Appendix B
Periodic Table..............................................................
165
Glossary .......................................................................
167
Index ............................................................................
173
ix
Tables and Figures
TABLES
2. 1.
The eight base unit s of SI .........................................
13
2. 2.
St andard pref ix of unit s........................................
15
2. 3.
Some indicat ors and t heir colour in acid or base
solut ions ...................................................................
23
pH value and it s corresponding Molar
concent rat ion ...........................................................
24
2. 5.
Examples of weak acids and t heir f ormula ..............
25
2. 6.
Examples of weak bases and t heir f ormula .............
25
2. 7.
pKa values of acids and bases usef ull f or buf f er
preparat ion ..............................................................
27
2. 8.
Biological buf f ers and t heir buf f ering range ...........
29
3. 1.
The wavelengt h of elect romagnet ic light s ..............
33
3. 2.
Visible colour and t he absorbed colour ...................
36
4. 1.
An example of calculat ion in t he det erminat ion of
t ot al cholest erol in serum sample ...........................
74
An example of calculat ion in t he det erminat ion of
HDL cholest erol in serum sample ............................
76
Dilut ion of P st ock solut ion t o obt ain a series of P
st andard concent rat ion ............................................
104
The amount of ml st andard solut ion and reagent
added in t he det erminat ion of P in a sample ..........
105
6. 3.
Preparat ion of blank solut ion ..................................
105
7. 1.
Dilut ion of st ock sol ut ion t o obt ain varying
concent rat ion of Calcium st andard solut ion ............
120
2. 4.
4. 2.
6. 1.
6. 2.
x
7. 2.
10. 1.
Summary of procedures t o obt ain a st andard curve
f or calcium det erminat ion by Spect rophot omet er ..
121
Preparat ion of st andard t annic acid solut ion in
glass t ubes ................................................................
150
FIGURES
2. 1.
New pH paper (lef t ) and it s box (right ) .....................
21
2. 2.
Colourless acid HCl (lef t ) af t er addit ion wit h
indicat or phenol red t urns int o red (right ) ................
22
Colourless acid NaOH (lef t ) af t er addit ion wit h
indicat or phenol red t urns int o yellow (right ) ..........
22
2. 3.
3. 1.
3. 2.
3. 3.
3. 4.
3. 5.
3. 6.
3. 7.
A whit e light passing a prism will emit a spect rum
of colour (colour band) which consist s of purple,
blue, green, yellow, and red (Whit e et al . , 1965;
Grolier, 1975) ............................................................
33
Blue solut ion passing bl ue light which is caught by
human eyes or inst rument .........................................
35
St andard curve, t he relat ionship bet ween light
absorbed by a solut ion and t he concent rat ion of a
solut ion ......................................................................
40
Non linear curve of t he relat ionship bet ween
absorbance and concent rat ion ..................................
41
Several t ypes of spect rophot omet ers’ cuvet t e; t op:
rect angular & oval like shape cuvet t es, bot t om:
micro-cuvet t es (Sigma, 2007) ....................................
42
Out line of a spect rophot omet er (modif ied f rom
Segel, 1975) .............................................................
43
Spect ronic 20 (Spect rophot omet er) .......................
44
xi
3. 8.
3. 9.
3. 10.
Spect ronic 20 (DIGITAL Spect rophot omet er f rom
Thermo Fisher Scient if ic, 2009) .............................
Spect ro UVS-2700 is a double-beam UV-VIS
Spect rophot omet er wit h eight cuvet t e holders
(Labomed, 2001) .....................................................
Spect rophot omet er UV-1201 (Shimadzu) wit h 6
cuvet t e holders at Nut rit ional Biochemist ry Lab.
where t he aut hor is af f iliat ed………............................
45
46
46
3. 11.
Spect ronic-20 wit h it s part s .......................................
47
3. 12.
UV-1201 Spect rophot omet er rear view (Shimadzu,
1994) .........................................................................
51
UV-1201 Spect rophot omet er rear view (Shimadzu,
1994) .........................................................................
51
Sample compart ment UV-1201 spect rophot omet er
(Shimadzu, 1994) ......................................................
52
3. 15.
Keypad f unct ions (Shimadzu, 1994) ..........................
53
4. 1.
Preparat ion of serum or plasma f rom whole blood ...
63
4. 2.
Serum is kept in eppendorf t ube ...............................
71
4. 3.
Procedures f or det erminat ion of t ot al cholest erol
3. 13.
3. 14.
(modified from DiaSys, 2006; Murwani and
Bayuardhi, 2007)........................................................
4. 4.
4. 5.
Lef t : t he colour of blank, st andard, and sample
solut ion bef ore reading in spect rophot omet er; right
: blank, st andard and sample in cuvet t e ready f or
absorbance reading ....................................................
xii
73
Procedures f or det erminat ion of HDLcholest erol
(modified from DiaSys, 2006; Murwani, 2008b) .........
4. 6.
72
Procedures
f or
det erminat ion
of
Triglyceride
77
(modified from DiaSys, 2006; Murwani, 2008b) .........
79
4. 7.
Fresh sample meat is weighed ...................................
83
4. 8.
Fresh sample meat is dispersed in NaCl solut ion ......
83
4. 9
Meat sample solut ion is dilut ed wit h NaCl ...............
83
4. 10.
St andard and sample af t er addit ion of cholest erol
reagent s .....................................................................
84
St andard and sample af t er addit ion of cholest erol
reagent s in cuvet t e ready f or absorbance reading ...
84
5. 1.
St andard prot ein solut ions .........................................
92
6. 1.
Amonium Molybdat e solut ion .....................................
98
6. 2.
ANS (Amino-Napht ol-Sulf onic Acid) Lef t : Clear
brown ANS solut ion. Right : ANS solut ion in a dark
brown bot t le …………………………………………………………….
4. 11.
6. 3.
99
Phophor st ock solut ion of 2000 ppm in volumet ric
f lask ............................................................................
103
A series of P st andard solut ion which are made f rom
dilut ion of st ock solut ion ...........................................
104
St andard solut ions t hat has been added wit h
reagent and developed int o color solut ion
Concent rat ion of st andard solut ion f rom lef t t o
right : 0. 5 ppm, 1 ppm, 2 ppm, 3 ppm, 4 ppm, 5
ppm, 6 ppm ................................................................
106
10. 1.
Folin Ciocalt eu reagent in dark bot t le ......................
148
10. 2.
St andard t annic acid solut ions in cuvet t e ready f or
absorbance reading (lef t : blank, second t o 6t h
cuvet t es t o t he right are st andards) .........................
6. 4.
6. 5.
10. 3.
Lef t : t he colour of sample solut ions bef ore reading
in spect rophot omet er; right : samples in cuvet t e
ready f or absorbance reading (sorghum sample) ......
150
151
xiii
C ha pte r I
Intro duc tio n to
Nutritio n Ana lysis
1
xiv
In human, nut rit ion is def ined as “ t he sci ence of f oods, t he
nut r i ent s and t he subst ances t her ei n, t hei r act i on, i nt er act i on
and bal ance i n r el at i on t o heal t h and di seases” . Anot her
nut rit ion def init ion is “ t he sum of t he pr ocesses i nvol ved i n
t he t aki ng i n of nut r i ent s and t hei r assi mi l at i on and use f or
pr oper body f unct i oni ng and mai nt enance of heal t h. The
successi ve st ages i ncl ude i ngest i on, di gest i on, absor pt i on,
assi mi l at i on, and excr et i on” (AHMD, 2007). In shor t Nut r i t i on
i s “ t he sci ence of f ood at wor k i n t he body” . Food f or ani mal s
or l i vest ock i s t er med as f eed . Theref ore, adopt ing t his
def init ion of nut rit ion f or animal, animal nut rit ion can be
def ined accordingly as t he sci ence of f eed at wor k i n t he body
of an ani mal . According t o WHO of t he Unit ed Nat ions, healt h
is f urt her def ined as “ A st at e of compl et e physi cal , ment al
and soci al wel l -bei ng and not mer el y t he absence of di sease
and i nf i r mi t y” . Animal nut rit ion deals wit h t he science of
f eed at work in t he body t o support opt imal product ivit y. The
product ivit y can be f urt her grouped according t o t he purpose
of t he animal being raised, i. e. as livest ock f or human
consumpt ion, as companion animals, sport s (such as race
horses), work (such as buf f alo in rice producing count ries), or
cont est . Product ivit y of livest ock animal s is dif f erent f rom
t hose f or work and ot her purposes. However, t hey all deserve
t o have proper nut rit ion in order t o achieve opt imal physical
and physiological development . Furt her, recent development
2
xv
xvi
C ha pte r II
Units o f a Nutrie nt
C o nc e ntra tio n
11
xvii
INTRODUCTION
In a st udy of provit amin A β-carot ene supplement at ion in a
diet , t he dist ribut ion in t he body, and t he act ivit y of t he
enzyme involved in β-carot ene met abolism in chicks,
a
publicat ion under Mat erials & Met hods described t he f ollowing
procedures (Murwani, 2002):
One-day-old male leghorn chicks were given a vitamin
A deprived diet ad libitum (Table 1). They were kept
under controlled light and warm temperature, and water
was provided on free access. After feeding the chicks
on vitamin-A deprived diet for 10 days to lower and
equalise hepatic retinol storage, 28 chicks with similar
average body weight were assigned into four treatment
groups to receive diet containing: 1) 20% gluten protein
supplemented with 1.5 µg β-carotene/g diet, 2) 20%
gluten protein supplemented with 15 µg β-carotene/g
diet, 3) 20% casein protein supplemented with 1.5 µg βcarotene/g diet, 4) 20% casein protein supplemented
with 15 µg β-carotene/g diet.
Anot her publicat ion (Murwani, 2001) described t he assay of an
enzyme involved in t he conversion of β-carot ene int o ret inol in
chick int est inal mucosa wit h t he f ollowing procedures :
ARAT was assayed by measuring the formation of
3
labeled [ H] retinyl palmitate after incubating radioactive
3
50,000 cpm [ H] retinol and 10 nmol unlabeled retinol
with unlabeled palmitoyl-CoA in the presence of
microsomal ARAT.
The assay procedure were
basically the same as that described by Helgerud et al.,
(1982, 1983), and modified so that radioactive retinol
12
xviii
xix
C ha pte r III
Princ iple s o f
Spe c tro pho to m e te r
31
xx
Nut rient analysis in f eed or f ood is an import ant st ep in
composing a good diet t o provide all nut rient s needed f or
normal growt h and development . It is also import ant in
st udying or t he f at e of nut rient s during met abolism as has
been described in Chapt er I. Macro and micro nut rient s in f eed
or
f ood
(animal
product
is part
of
f ood
f or
human
consumpt ion), or in cert ain part s of body organs can be
det ermined qualit at ively or quant it at ively by various met hods.
Quant it at ive analyses can be done wit h t he aid of analyt ical
equipment called Spect rophot omet er. Spect rophot omet er is
an analyt ical inst rument which works on t he basis of simpl e
colourimet ric principles i. e. absorbance of light by colour
solut ion. Thus t his chapt er begins wit h an underst anding of
t he
light
and
colourimet er
principles.
Consequent ly,
spect rophot omet ry as t he science of spect rum of a compound
and as t he basis of quant it at ive det erminat ion of a nut rient
has a broad and growing applicat ion.
WHITE LIGHT / SUNLIGHT
Light t hat is emit t ed by t he sun and seen by human eyes as
whit e light consist s of various colours. If a beam of light
passes t hrough a prism, a range of colour light (purple, blue,
green, yellow, and red) will be emit t ed f rom t he prism (see
Figure 3. 1).
Light is an elect romagnet ic radiat ion which consist s of bands
of cert ain wavelengt hs and so does sunlight which
32
xxi
xxii
C ha pte r IV
De te rm ina tio n o f Lipid
In Se rum a nd Tissue
xxiii
61
To st udy t he ef f ect of f reshly made garlic powder on t he
serum concent rat ion of lipid in broilers, a publicat ion under
Mat erials & Met hods described t he f ollowing procedures :
One hundred male Ross 308 chicks were divided
into two equal groups. Group 1 received regular
broiler diet supplemented with 2% garlic powder.
Group 2 received regular broiler diet. All chickens
were slaughtered on day 50, every carcass was
weighed and its adipose tissue content was
determined. At the time of slaughtering, a 5-ml
blood sample was collected into a test tube from
each chicken. Tubes were labelled and then
centrifuged at 3,000 RPM for 10 min to collect blood
serum samples. The sera were analyzed to
measure total cholesterol and total triglyceride using
enzymatic colorimetric methods (Dehkordi et al.,
2009).
We can not e f rom t he above procedure t hat blood serum
samples were collect ed and t he concent rat ion of
t ot al
cholest erol and t ryglyceride were det ermined by colorimet ric
met hod or in anot her word by spect rophot omet ry t hat has
been discussed in Chapt er 2. The f irst st ep in det erminat ion of
cholest erol in serum is preparat ion of serum samples f rom
blood wit hdrawn f rom t he chicken or ot her experiment al
animals such as mouse, rat s, or ot her experiment al livest ock.
xxiv
62
C ha pte r V
De te rm ina tio n o f
Pro te in in Blo o d Se rum
87
xxv
To st udy t he act ivit y of t he enzyme involved in β-carot ene
met abolism in chicks f ollowing supplement at ion wit h nat ural
β-carot ene, a publicat ion under Mat erials & Met hods described
t he f ollowing procedures (Murwani, 2002):
Protein concentration in intestinal mucosa preparation
was measured following Lowry method using Bovine
Serum Albumin as standard. The same microsome
preparation was assayed for the enzyme activity which
catalysed the esterification of retinol (as the result of βcarotene splitting) into retinyl ester.
The above procedures described t he det erminat ion of prot ein
concent rat ion
of
int est inal
mucosa
preparat ion.
This
det erminat ion is needed t o express t he act ivit y of enzyme in
Unit per mg prot ein. Anot her mat erial and met hod can
describe t he det erminat ion of prot ein concent rat ion in serum
in order t o see normalit y or abnormalit y which might occur
f ollowing cert ain nut rient t reat ment .
Prot eins make up 6–8% of t he bl ood. The maj orit y of prot ein
f ound in serum consist of albumin, globulin, f ibrinogen and
prot hrombin. Normal prot ein in chicken serum is 3-3. 3 g/ dl,
and in human plasma is 7-7. 5 g/ dL. Prot ein in blood carry a
variet y of import ant f unct ions i. e. circulat ory, prot ect ion, and
regulat ion. As circulat ory f unct ion it t ransport s biomolecules
such as carbohydrat e, lipid, prot ein, vit amins, minerals,
hormones,
enzymes,
complement
component s,
prot ease
inhibit ors, et c. As regulat or, it regulat es pH, t emperat ure and
osmot ic balance. As
88
xxvi
xxvii
C ha pte r VI
De te rm ina tio n o f
Pho spho r in Fe e d o r
Fo o d
xxviii
95
A st udy was conduct ed t o evaluat e t he ef f icacy of 25hydroxycholecalcif erol
[ 25-(OH)D3]
to
minimize
t he
development of t ibial dyschondroplasia (TD) and improve
phyt at e phosphorus ret ent ion in Ross cockerels during t he
st art er
period
(Ledwaba and
Roberson,
2003).
Part
of
mat erials and met hods was described below.
A total of 240 1-d-old male broiler chicks were used for
each experiment. Ten chicks per pen were assigned
randomly to each of 24 pens in an electrically heated
battery brooder housed in a room without windows.
Room temperature was maintained at approximately
23°C . Feed and water were provided ad libitum in all
experiments.
The compositions of the corn-soybean
meal based diets are listed in Table 1.
Dietary 25-
(OH)D3 premix was donated and delivered from
commercial mill. Chromix oxide was used as an
external indicator at 0.10% of the basal diet to
determine
phytate
phosphorus
retention.
Feed
phosphorus was determined colorimetrically with a
spectrophotometer using the method described by
Gomori (1942).
In t he mat erial and met hod it is writ t en t hat det erminat ion of
phosphor in f eed was done by spect rophot omet ry.
Phosphor is a macro mineral essent ial f or bone f ormat ion.
Phosphor
combines
wit h
calcium
to
f orm
calcium
hydroxyapat it e which is t he const it uent of bone and t eet h.
96
xxix
xxx
Chapter VII
Determination of
Calcium
115
xxxi
A st udy was conduct ed t o evaluat e t he ef f icacy of 25hydroxycholecalcif erol
[ 25-(OH)D3]
to
minimize
t he
development of t ibial dyschondroplasia (TD) and improve
phyt at e phosphorus ret ent ion in Ross cockerels during t he
st art er period (Ledwaba and Roberson, 2003). Part of mat erials
and met hods is described below :
Blood samples
were obtained at 13 d of age via
cardiac puncture from one chick per pen and were
analyzed for each individual chick picked randomly as a
representative of the pen. The blood was centrifuged
for 15 min at 3,000 x g to extract serum and analyzed
for serum phosphorus (Goldenberg and Fernandez,
1966) and calcium (Moorhead and Biggs, 1974)
concentrations using the same spectrophotometer as
for feed phosphorus analysis.
Calcium is one of macro minerals which plays a cent ral role in
bone f ormat ion and maint anance in animals and human. In
chicken, 94% of egg shell is composed of calcium carbonat e.
When diet ary calcium is not suf f icient it can lead t o a
decrease in egg product ion, weight , and specif ic gravit y, f eed
consumpt ion, body weight , bone densit y and st rengt h. Diet ary
calcium t heref ore is essent ial f or f ast growing birds such as
broilers and egg producing birds such as layers. In layers,
t here is a high demand f or calcium during egg product ion.
When t his demand is not met it can lead t o t he occurence of
116
xxxii
xxxiii
C ha pte r VIII
De te rm ina tio n o f DNA
a nd RNA fro m C e lls o r
Tissue o f a n Anim a l
xxxiv
123
A st udy was conduct ed t o invest igat e t he development al
relat ionship bet ween f at t y acid composit ion in dif f erent lipid
f ract ions and st earoyl-CoA desat urase (SCD) gene expression in
st eer muscles during growt h. Under Mat erials & Met hods part
of t he procedures is described below :
Twenty male Korean Hanwoo steers were used in this
study. Steers were slaughtered at 6, 12, 18, 24 and 30months old, respectively. Muscle samples were taken
th
from longissimus dorsi between 5
vertebrae of steers.
th
and 6
lumbar
All samples were immediately
frozen in liquid nitrogen and stored at -80°C until
analysis.
Total RNA was isolated from loin muscle
using Trizol reagent (Life Technologies Inc., Grand
Island, NY, USA) according to the manufacturer’s
instructions. Following RNA isolation, the concentration
and purity of the prepared RNA were analyzed by
optical density measurements at 260/280 nm (Lee et
al., 2005).
The mat erials and met hods above showed t hat RNA was
det ermined by opt ical densit y measurement . Opt ical densit y
or abbreviat ed as OD is t he same as absorbance and indicat es
t hat it is measured spect rophot omet rically (See Chapt er 2).
DNA st ands f or Deoxyribo Nucleic Acid and RNA st ands f or Ribo
Nucleic Acid. DNA and RNA are t he genet ic codes t hat
det ermine t he charact erist ic of an individual. Nucleic acids
are f ound in t he nucleus of animals, plant s, and microbial
124
xxxv
xxxvi
C ha pte r IX
De te rm ina tio n o f
Enzym e Ac tivity
133
xxxvii
A st udy was conduct ed t o measure t he act ivit y of int est inal
phyt ase act ivit y in broiler chickens as af f ect ed by diet ary
calcium and vit amin D. Under mat erials and met hods t he
f ollowing procedures were described:
Hubbard x Peterson, male chicks were fed diets with or
without 0.21 mg/kg 25-OH D3 with varying dietary Ca
concentrations (4 or 9 g/kg) supplied primarily from
CaCO3 from 7 to 21 d of age (five pens/diet, three
birds/pen) in brooder battery cages. All birds were fed
diets calculated to contain 0.20% nonphytate P. Diets
contained Celite as an inert filler and as a digestive
marker at a minimum of 10 g/kg diet to allow for
determination of apparent phytate phosphorous (PP)
hydrolysis. Dietary Ca and total P were determined by
inductively
coupled
plasma-emission
spectroscopy
(ICP). Phytate-P content of diets was determined as
described below. At 7 d of age, birds were weighed
individually and allocated to experimental pens, such
that BW differences were minimized. At 21 d of age,
birds were euthanized by cervical dislocation.
The
duodenum and jejunum were rinsed in ice-cold saline,
and the mucosa was scraped and frozen (-80°C) for
future isolation of brush-border vesicles (BBV) and
subsequent
intestinal
phytase
determinations
(Applegate et al., 2003)
Enzymes are prot ein
t hat
speed
biochemical
react ions.
Enzymes have many applicat ions in t he f ield of animal and
134
xxxviii
xxxix
C ha pte r X
De te rm ina tio n o f To ta l
Phe no lic C o m po und
xl145
Polyphenols or phenolics are groups of subst ance nat urally
f ound in t he plant kingdom wit h chemical charact erist ics of
having aromat ic ring wit h one or more hydroxyl groups. They
are locat ed in t he vacuole and t end t o be wat er-soluble as
t hey are f ound in associat ion wit h sugars.
Included in
polyphernols are 1) simple phenols and t heir derivat ives such
as cat echol, eugenol, hydroxycinnamic acids (p-coumaric,
caf f eic,
f erulic
and
sinapic
acids),
and
coumarins,
2)
f lavonoids such as f lavonols and f lavones, chalcone and aurone
pigment s, f lavanones, and isof lavonoids, 3) ant hocyanins, 4)
Flavan-3-ols and f lavan-3, 4-diols, 5)Tannins which consist of
hydrolysable
t annins
and
condensed
t annins
(proant o-
cyanidins) (Scalbert and Williamson, 2000; Bennick, 2002).
Polyphenols can
phyt onut rient
excert
and
as
dual
ef f ect
i. e.
ant inut rient .
as f unct ional
As
f unct ional
phyt onut rient , many polyphenols have been shown t o have
ant ioxidant
propert ies which are import ant
syst em. The ant ioxidant propert ies of
in biological
isol at ed or pure
polyphenols are used t o enrich f eed and f ood t o improve
nut rit ional st at us of animals and human. Nat urally occurring
poliphenol in f eed mat erials such as sorghum can also be used
(Murwani, 2008a, b, c). The ant i-nut rient ef f ect of nat urally
occurring poliphenolic compounds of sorghum is well known. It
is det riment al t o poult ry when given in large amount s (Nyacot i
et al. , 1996). However, when it is used in small amount in t he
diet , it can exert it s biological f unct ion as immunomodulat or
146
xli
xlii
Re fe re nc e s
Alexander, R. R. , Grif f it hs, J. M. and Wilkinson, M. L. 1984. Basic
Biochemical Met hods. John Wiley & Sons Inc. New York.
AHMD. 2007. The American Herit age® Medical Dict ionary. Hought on
Mif f lin Company.
AOAC.
2000. Of f icial Met hods of Analysis. Associat ion of Of f icial
Analyt ical Chemist . Inc. , Vi rginia. USA.
AOAC. 1984. Of f icial Met hods of Analyses. Associat ion of Of f icial
Analyt ical Chemist s. Inc. , Arlingt on. USA.
Applegat e, T. J. , Angel , R. , and Classen, H. L. 2003. Ef f ect of
diet ary calci um, 25-hydroxycholecalcif erol , or bird st rain
on small int est inal phyt ase act ivit y in broiler chi ckens.
Poult ry Sci ence 82: 1140-1148.
Bennick, A. 2002. Int eract ion of plant polyphenols wit h salivary
prot eins. Crit ical Reviews in Oral Biology and Medicine.
13(2): 184-196
Bensadoun, A. and Rot hf iled, A. 1972. The f orm of absorpt ion of
lipids in t he chicken, Gal l us domest icus. Proceedi ngs of
t he Societ y f or Experiment al Biology and Medicine 41: 814817.
Bohac, C. E. , Rhee, K. S. , Cross, H. R. and Ono, K. 1984. Assessment
of met hodologies f or col orimet ric cholest erol assay of
meat . Journal Food Science 53(6): 1642-1693.
Coles, E. H. 1986. Vet erinary Cli nical Pat hology. 4t h Ed. W. B.
Saunders Company, Phi ladelphia.
Dehkordi, S. H. , Moghadam, A. Z. , Maghsoudi, N. , Aali, E. , Gerami,
R. , and Dehsadeghi, E. 2009. The ef f ect s of f resh garlic on
t he serum concent rat ion of t ot al cholest erol , t ot al
t riglyceride and adipose t issues of broi lers. Comparat i ve of
Clinical Pat hology. Published on line 25 Agust us 2009.
DiaSys. 2006. Technical Sheet of Triglyceride, Cholest erol and HDLCholest erol . DiaSys Diagnost ic Syst ems GmbH. Germany.
Dorey, R. C. , and Draves, J. 1998. Quant it at ive Anal ysis Laborat ory:
A New Approach.
Universit y of Cent ral Arkansas,
Depart ement of Chemist ry. Conwey. Arkansas.
157
xliii
xliv
APPENDIX A
Ato m ic We ig hts o f
The Ele m e nts
163
xlv
ATOMIC WEIGHTS OF THE ELEMENTS
Element
Symbol
Aluminum
Ant imony
Argon
Arsenic
Barium
Beryllium
Bismut h
Boron
Bromine
Cadmium
Calcium
Carbon
Cerium
Cesium
Chlorine
Chromium
Cobalt
Copper
Dysprosium
Erbium
Europium
Fluorine
Gadolinium
Gallium
Germanium
Gold
Haf nium
Helium
Holmium
Hydrogen
Indium
Iodine
Iridium
Iron
Krypt on
Lant hanum
Lead
Lit hium
Lut ecium
Magnesium
Manganese
Mercury
Molybdenum
Al
Sb
Ar
As
Ba
Be
Bi
B
Br
Cd
Ca
C
Ce
Cs
Cl
Cr
Co
Cu
Dy
Er
Eu
F
Gd
Ga
Ge
Au
Hf
He
Ho
H
In
I
Ir
Fe
Kr
La
Pb
Li
Lu
Mg
Mn
Hg
Mo
xlvi
164
At omic
weight
26. 98
121. 75
39. 95
74. 92
137. 34
9. 01
208. 98
10. 81
79. 90
112. 40
40. 08
12. 01
140. 12
132. 91
35. 45
52. 00
58. 93
63. 55
162. 50
167. 26
151. 96
19. 00
157. 25
69. 72
72. 59
196. 97
178. 49
4. 00
164. 93
1. 008
114. 82
126. 91
192. 22
55. 84
83. 80
138. 91
207. 21
6. 94
174. 97
24. 31
54. 94
200. 59
95. 94
Element
Symbol
Neodymium
Neon
Nickel
Niobium
Nit rogen
Osmium
Oxygen
Palladium
Phosphorus
Plat inum
Pot assium
Praseodymium
Prot act inium
Radium
Radon
Rhenium
Rhodium
Rubidium
Rut henium
Samarium
Scandium
Selenium
Silicon
Silver
Sodium
St rot ium
Sulf ur
Tant alum
Tellurium
Terbium
Thallium
Thorium
Thulium
Tin
Tit anium
Tungst en
Uranium
Vanadium
Xenon
Yt t erbium
Yt t rium
Zinc
Zirconium
Nd
Ne
Ni
Nb
N
Os
Ni
Nb
N
Os
O
Pd
P
Pt
K
Pr
Pa
Ra
Rn
Re
Rh
Rb
Ru
Sm
Sc
Se
Si
Ag
Na
Sr
S
Ta
Te
Tb
Ti
W
U
V
Xe
YB
Y
Zn
Zr
At omic
weight
144. 27
20. 18
58. 71
92. 91
14. 01
190. 2
16. 00
106. 4
30. 97
195. 09
39. 09
140. 91
231. 04
226. 03
222
186. 2
102. 91
85. 47
101. 07
150. 43
44. 96
78. 96
28. 09
107. 87
23. 00
87. 62
32. 06
180. 95
127. 60
158. 93
204. 39
232. 04
169. 93
118. 69
47. 90
183. 85
238. 03
50. 94
131. 30
173. 04
88. 91
65. 38
91. 22
APPENDIX B
Pe rio dic Ta b le
xlvii
165
166
xlviii
PERIODIC TABLE OF THE ELEMENTS
(IUPAC, 2008)
1
2
H
1.0079
3
III
13
II
2
4
5
IV
14
6
V
15
7
VI
16
8
9
Li
Be
B
C
N
O
6.941
11
9.0122
12
10.811
13
12.011
14
14.007
15
15.999
16
Na
Mg
3
4
22.990
19
24.305
20
K
Ca
Sc
Ti
39.098
37
40.078
38
44.956
39
47.88
40
Rb
Sr
85.468
87.62
55
56
21
Y
88.906
57-71
22
5
23
V
50.942
41
Zr
Nb
91.224
72
92.906
73
6
24
7
25
8
26
9
27
10
28
11
29
Cr
Mn
Fe
Co
Ni
Cu
51.996
42
54.938
43
55.847
44
58.933
45
58.69
46
63.546
47
Mo
95.94
74
Tc
{98}
75
12
30
Zn
65.39
48
Al
Si
26.982
31
28.086
32
Ga
69.723
49
Ge
72.61
50
P
30.974
33
As
74.922
51
S
32.066
34
Se
78.96
52
Ru
Rh
Pd
Ag
Cd
In
Sn
Sb
Te
101.07
76
102.91
77
105.42
78
107.87
79
112.41
80
114.82
81
118.71
82
121.75
83
127.60
84
Tl
204.38
Hf
Ta
W
Re
180.95
183.85
186.21
89-103
104
105
106
Fr
Ra
**Ac
Unq
Unp
Unh
At om i c w ei ght s ar e based on 12C = 12 and confor m t o t he 1987 IUPAC r epor t val ues r ounded t o 5
si gni fi cant di gi t s. Num ber s i n { } i ndi cat e t he m ost st abl e i sot ope.
226.03
227.03
{261}
{262}
{263}
57
58
59
60
64
65
66
67
207.2
68
Bi
208.98
69
Po
I
126.90
85
178.49
63
Pb
Kr
83.80
54
*La
** Actinides
Hg
200.59
Br
79.904
53
138.91
62
Au
196.97
Ar
39.948
36
Ba
61
Pt
195.08
{209}
70
Ne
20.180
18
Cl
137.33
88
* Lanthanides
Ir
He
4.0026
10
35.453
35
132.91
87
190.2
192.22
F
18.998
17
Cs
{223}
Os
VII
17
At
{210}
71
*La
Ce
Pr
Nd
Pm
Sm
Eu
Gd
Tb
Dy
Ho
Er
Tm
Yb
Lu
138.91
140.12
140.91
144.24
{145}
150.36
151.97
157.25
158.93
162.50
164.93
167.26
168.93
173.04
174.97
89
90
91
92
93
94
97
98
99
100
101
102
103
95
96
**Ac
Th
Pa
U
Np
Pu
Am
Cm
Bk
Cf
Es
Fm
Md
No
Lr
227.03
232.04
231.04
238.03
237.05
{244}
{243}
{247}
{247}
{251}
{252}
{257}
{258}
{259}
{262}
Xe
131.29
86
Rn
{222}
G LO SSARY
AAS
:
At omic Aborpt ion Spect rophot omet er,
an analyt ical inst rument which ionizes
mineral sample and t he absorpt ion of
t he
ionized
at om
is
measured
spect rophot omet rically.
Abdominal fat
:
Fat which is deposit ed in t he abdominal
cavit y of broilers or commercial meat
chicken.
Albumin
:
A t ype of prot ein t hat is soluble in
wat er and in wat er half sat urat ed wit h
a salt such as ammonium sulf at e. Serum
albumin is a component of blood serum.
Egg and milk also cont ain albumin.
Seeds cont ain very small amount s of
albumins.
Antibody
:
Prot ein which is secret ed by specialized
immune cells i. e. B cells. It prot ect s
t he body against f oreign ant igen.
Antibody titers
:
The quant it y of ant ibody produced by B
cells in response t o ant igen.
Antigen
:
Spesif ic
molecules
of
f oreign
microorganism or cells or t issue.
Aqueous phase
:
liquids t hat are wat er-soluble. Because
t hey mix wit h wat er, t hey f orm a liquid
phase which are not separable. For
example, met hanol is mixed wit h
wat er, t heref ore t hey do not f orm a
separat e phase.
Arachidonic
acid
:
is a carboxylic acid. Arachidonic acid is
import ant because t he human body
uses it as a st art ing mat erial in t he
167
xlix
l
Inde x
A
AAS, 9, 117, 118, 167
Abdominal f at , 69, 136, 139, 167
absorbance, 32, 37-41, 43, 45-47, 49, 50, 56, 58, 59, 73, 74,
76, 79, 81, 84, 91-93, 106, 107, 109, 120, 121, 124, 132,
137, 138, 141, 143, 149-151, 154, 155, 168.
acet ic acid, 25, 27-29, 64, 81, 91, 110, 118, 136
acid, 5-7, 13, 19-30, 64, 65, 67, 68, 81, 91, 96, 98, 99, 110,
118, 124, 125, 132, 136, 137, 141, 146-152, 154
acidit y, 21, 25
ad l i bi t um , 12, 96
addit ives, 129
Adenosine Tri Phosphat e (ATP), 7
adipocyt es, 68
Adipose, 62, 68
ADP, 96
agarose, 5
alanine, 140-142
ALAT, 140-142
albumen, 82
alkaline, 89, 131
alkalinit y, 21, 25
amino acid, 5, 28, 141
aminohexanoic acid, 13, 19
Amino-Napht ol-Sulf onic Acid, 98, 99, 110
ammonia, 25, 26, 135
ammonium molybdat e, 107, 112
ammonium sulf at e, 155, 167
ammonium vanadat e, 98, 107, 137
ampere, 13
animal welf are, 3
ant ibiot ic, 17, 138, 142-144, 160
ant ibody, 5, 89, 147
ant icoagulant , 64
173
li
lii
liii
TEC HNIQ UES O F ANALYSES IN
NUTRITIO NAL BIO C HEMISTRY
ii
iii
TEC HNIQ UES O F ANALYSES IN
NUTRITIO NAL BIO C HEMISTRY
Retno Murwani
Head of Labor at or y of Nut r it ional Biochemist r y
Dept . of Nut r it ion and Feed Science
Facul t y of Ani mal Science
Diponegor o Univer sit y Semar ang
Published by BP Undip
Semarang
November 2009
iv
Book : Techniques of Analyses in Nut rit ional Biochemist ry; Ret no
Murwani (aut hor); 1st Ed; Badan Penerbit Undi p Semarang; 2009
xii + 184 pages; 16. 5 x 23. 5 cm
ISBN : 978-979-704-838-9
Tit le:
TEC HNIQ UES O F ANALYSES IN
NUTRITIO NAL BIO C HEMISTRY
Ret no Murwani
copyright 2009
Al l r ight s r eser ved
No part of t his book may be reproduced in any f orm, by mimeograph
or any ot her means, wit hout permission in writ ing f rom t he aut hor
v
Preface
This book is writ t en t o document empirical experience during
t he course of various research grant s t hat t he aut hor has
received and carried out , as well as t o document experience
of more t han 23 years of t eaching as a lect urer. This book is
writ t en as part of World Class Universit y Program f or Book
Writ ing launched by Diponegoro Universit y in t he year 2009.
The aut hor is one of t welve universit y lect urers who are
assigned t o writ e t he book in English language (SK Rekt or
No. 520/ SK/ H7/ 2009).
This book is writ t en t o help st udent s and ot her readers in
carrying analyses eit her on t heir own, or as a ref erence and
guide in underst anding analyt ical met hods writ t en in scient if ic
j ournals t o support t heir research. The book consist s of t en
chapt ers, covering several maj or and minor nut rient s as well
as phyt onut rient such as polyphenol. It is beyond t he scope of
t his book t o cover all nut rient s known t oday. However, t he
principles and t echniques can be applied t o almost any
nut rient s. Each chapt er is int roduced wit h a paragraph t hat
gives a sample phrase which can ref lect t he need of a nut rient
analysis in t he cont ext of a scient if ic research t hat has been
published in scient if ic j ournals, or on a nut rient label of f eed
or f ood product s. This int roduct ion is meant t o ease t he
vi
reader t o relat e t o inf ormat ion or an inst ruct ion f ound in
many published works wit h t he mat erials given in each
chapt er. Af t er reading t his book t he readers wil l hopef ully be
able t o underst and writ t en analyt ical procedures and execut e
t he procedures int o pract ice in nut rit ion analyses.
It is my hope t hat t his book will be usef ul not only f or st udent s
in animal science f acult y, but also f or ot her st udent s and
readers who need a guide t o carry out nut rient analyses.
Grat ef ul acknowledgement is due t o t he f inancial support
f rom World Class Universit y Program f or
Book Writ ing,
Diponegoro Universit y, year 2009, and Prof . Dr. Ret mono, who
is a Prof essor in English Educat ion, as t he appoint ed expert f or
t he proof reading of t he English language.
Aut hor
Ret no Murwani
November, 2009
vii
Contents
Pref ace ........................................................................
v
Cont ent s ......................................................................
vii
Tables and Figures.......................................................
ix
I
Int roduct ion t o Nut rit ion Analysis................................
1
II
Unit s of A Nut rient Concent rat ion...............................
11
III
Principles of Spect rophot omet er .................................
31
IV
Det erminat ion of Lipid in Serum and Tissue ...............
61
V
Det erminat ion of Prot ein in Blood Serum ....................
87
VI
Det erminat ion of Phosphor in Feed or Food ................
95
VII
Det erminat ion of Calcium ...........................................
115
VIII
Det erminat ion of DNA and RNA From Cells or Tissue
123
of an Animal ...............................................................
IX
Det erminat ion of Enzyme Act ivit y...............................
133
X
Det erminat ion of Tot al Phenolic Compound ................
145
Ref erences ...................................................................
157
Appendix A
At omic Weight s of The Element s ………………………………
viii
163
Appendix B
Periodic Table..............................................................
165
Glossary .......................................................................
167
Index ............................................................................
173
ix
Tables and Figures
TABLES
2. 1.
The eight base unit s of SI .........................................
13
2. 2.
St andard pref ix of unit s........................................
15
2. 3.
Some indicat ors and t heir colour in acid or base
solut ions ...................................................................
23
pH value and it s corresponding Molar
concent rat ion ...........................................................
24
2. 5.
Examples of weak acids and t heir f ormula ..............
25
2. 6.
Examples of weak bases and t heir f ormula .............
25
2. 7.
pKa values of acids and bases usef ull f or buf f er
preparat ion ..............................................................
27
2. 8.
Biological buf f ers and t heir buf f ering range ...........
29
3. 1.
The wavelengt h of elect romagnet ic light s ..............
33
3. 2.
Visible colour and t he absorbed colour ...................
36
4. 1.
An example of calculat ion in t he det erminat ion of
t ot al cholest erol in serum sample ...........................
74
An example of calculat ion in t he det erminat ion of
HDL cholest erol in serum sample ............................
76
Dilut ion of P st ock solut ion t o obt ain a series of P
st andard concent rat ion ............................................
104
The amount of ml st andard solut ion and reagent
added in t he det erminat ion of P in a sample ..........
105
6. 3.
Preparat ion of blank solut ion ..................................
105
7. 1.
Dilut ion of st ock sol ut ion t o obt ain varying
concent rat ion of Calcium st andard solut ion ............
120
2. 4.
4. 2.
6. 1.
6. 2.
x
7. 2.
10. 1.
Summary of procedures t o obt ain a st andard curve
f or calcium det erminat ion by Spect rophot omet er ..
121
Preparat ion of st andard t annic acid solut ion in
glass t ubes ................................................................
150
FIGURES
2. 1.
New pH paper (lef t ) and it s box (right ) .....................
21
2. 2.
Colourless acid HCl (lef t ) af t er addit ion wit h
indicat or phenol red t urns int o red (right ) ................
22
Colourless acid NaOH (lef t ) af t er addit ion wit h
indicat or phenol red t urns int o yellow (right ) ..........
22
2. 3.
3. 1.
3. 2.
3. 3.
3. 4.
3. 5.
3. 6.
3. 7.
A whit e light passing a prism will emit a spect rum
of colour (colour band) which consist s of purple,
blue, green, yellow, and red (Whit e et al . , 1965;
Grolier, 1975) ............................................................
33
Blue solut ion passing bl ue light which is caught by
human eyes or inst rument .........................................
35
St andard curve, t he relat ionship bet ween light
absorbed by a solut ion and t he concent rat ion of a
solut ion ......................................................................
40
Non linear curve of t he relat ionship bet ween
absorbance and concent rat ion ..................................
41
Several t ypes of spect rophot omet ers’ cuvet t e; t op:
rect angular & oval like shape cuvet t es, bot t om:
micro-cuvet t es (Sigma, 2007) ....................................
42
Out line of a spect rophot omet er (modif ied f rom
Segel, 1975) .............................................................
43
Spect ronic 20 (Spect rophot omet er) .......................
44
xi
3. 8.
3. 9.
3. 10.
Spect ronic 20 (DIGITAL Spect rophot omet er f rom
Thermo Fisher Scient if ic, 2009) .............................
Spect ro UVS-2700 is a double-beam UV-VIS
Spect rophot omet er wit h eight cuvet t e holders
(Labomed, 2001) .....................................................
Spect rophot omet er UV-1201 (Shimadzu) wit h 6
cuvet t e holders at Nut rit ional Biochemist ry Lab.
where t he aut hor is af f iliat ed………............................
45
46
46
3. 11.
Spect ronic-20 wit h it s part s .......................................
47
3. 12.
UV-1201 Spect rophot omet er rear view (Shimadzu,
1994) .........................................................................
51
UV-1201 Spect rophot omet er rear view (Shimadzu,
1994) .........................................................................
51
Sample compart ment UV-1201 spect rophot omet er
(Shimadzu, 1994) ......................................................
52
3. 15.
Keypad f unct ions (Shimadzu, 1994) ..........................
53
4. 1.
Preparat ion of serum or plasma f rom whole blood ...
63
4. 2.
Serum is kept in eppendorf t ube ...............................
71
4. 3.
Procedures f or det erminat ion of t ot al cholest erol
3. 13.
3. 14.
(modified from DiaSys, 2006; Murwani and
Bayuardhi, 2007)........................................................
4. 4.
4. 5.
Lef t : t he colour of blank, st andard, and sample
solut ion bef ore reading in spect rophot omet er; right
: blank, st andard and sample in cuvet t e ready f or
absorbance reading ....................................................
xii
73
Procedures f or det erminat ion of HDLcholest erol
(modified from DiaSys, 2006; Murwani, 2008b) .........
4. 6.
72
Procedures
f or
det erminat ion
of
Triglyceride
77
(modified from DiaSys, 2006; Murwani, 2008b) .........
79
4. 7.
Fresh sample meat is weighed ...................................
83
4. 8.
Fresh sample meat is dispersed in NaCl solut ion ......
83
4. 9
Meat sample solut ion is dilut ed wit h NaCl ...............
83
4. 10.
St andard and sample af t er addit ion of cholest erol
reagent s .....................................................................
84
St andard and sample af t er addit ion of cholest erol
reagent s in cuvet t e ready f or absorbance reading ...
84
5. 1.
St andard prot ein solut ions .........................................
92
6. 1.
Amonium Molybdat e solut ion .....................................
98
6. 2.
ANS (Amino-Napht ol-Sulf onic Acid) Lef t : Clear
brown ANS solut ion. Right : ANS solut ion in a dark
brown bot t le …………………………………………………………….
4. 11.
6. 3.
99
Phophor st ock solut ion of 2000 ppm in volumet ric
f lask ............................................................................
103
A series of P st andard solut ion which are made f rom
dilut ion of st ock solut ion ...........................................
104
St andard solut ions t hat has been added wit h
reagent and developed int o color solut ion
Concent rat ion of st andard solut ion f rom lef t t o
right : 0. 5 ppm, 1 ppm, 2 ppm, 3 ppm, 4 ppm, 5
ppm, 6 ppm ................................................................
106
10. 1.
Folin Ciocalt eu reagent in dark bot t le ......................
148
10. 2.
St andard t annic acid solut ions in cuvet t e ready f or
absorbance reading (lef t : blank, second t o 6t h
cuvet t es t o t he right are st andards) .........................
6. 4.
6. 5.
10. 3.
Lef t : t he colour of sample solut ions bef ore reading
in spect rophot omet er; right : samples in cuvet t e
ready f or absorbance reading (sorghum sample) ......
150
151
xiii
C ha pte r I
Intro duc tio n to
Nutritio n Ana lysis
1
xiv
In human, nut rit ion is def ined as “ t he sci ence of f oods, t he
nut r i ent s and t he subst ances t her ei n, t hei r act i on, i nt er act i on
and bal ance i n r el at i on t o heal t h and di seases” . Anot her
nut rit ion def init ion is “ t he sum of t he pr ocesses i nvol ved i n
t he t aki ng i n of nut r i ent s and t hei r assi mi l at i on and use f or
pr oper body f unct i oni ng and mai nt enance of heal t h. The
successi ve st ages i ncl ude i ngest i on, di gest i on, absor pt i on,
assi mi l at i on, and excr et i on” (AHMD, 2007). In shor t Nut r i t i on
i s “ t he sci ence of f ood at wor k i n t he body” . Food f or ani mal s
or l i vest ock i s t er med as f eed . Theref ore, adopt ing t his
def init ion of nut rit ion f or animal, animal nut rit ion can be
def ined accordingly as t he sci ence of f eed at wor k i n t he body
of an ani mal . According t o WHO of t he Unit ed Nat ions, healt h
is f urt her def ined as “ A st at e of compl et e physi cal , ment al
and soci al wel l -bei ng and not mer el y t he absence of di sease
and i nf i r mi t y” . Animal nut rit ion deals wit h t he science of
f eed at work in t he body t o support opt imal product ivit y. The
product ivit y can be f urt her grouped according t o t he purpose
of t he animal being raised, i. e. as livest ock f or human
consumpt ion, as companion animals, sport s (such as race
horses), work (such as buf f alo in rice producing count ries), or
cont est . Product ivit y of livest ock animal s is dif f erent f rom
t hose f or work and ot her purposes. However, t hey all deserve
t o have proper nut rit ion in order t o achieve opt imal physical
and physiological development . Furt her, recent development
2
xv
xvi
C ha pte r II
Units o f a Nutrie nt
C o nc e ntra tio n
11
xvii
INTRODUCTION
In a st udy of provit amin A β-carot ene supplement at ion in a
diet , t he dist ribut ion in t he body, and t he act ivit y of t he
enzyme involved in β-carot ene met abolism in chicks,
a
publicat ion under Mat erials & Met hods described t he f ollowing
procedures (Murwani, 2002):
One-day-old male leghorn chicks were given a vitamin
A deprived diet ad libitum (Table 1). They were kept
under controlled light and warm temperature, and water
was provided on free access. After feeding the chicks
on vitamin-A deprived diet for 10 days to lower and
equalise hepatic retinol storage, 28 chicks with similar
average body weight were assigned into four treatment
groups to receive diet containing: 1) 20% gluten protein
supplemented with 1.5 µg β-carotene/g diet, 2) 20%
gluten protein supplemented with 15 µg β-carotene/g
diet, 3) 20% casein protein supplemented with 1.5 µg βcarotene/g diet, 4) 20% casein protein supplemented
with 15 µg β-carotene/g diet.
Anot her publicat ion (Murwani, 2001) described t he assay of an
enzyme involved in t he conversion of β-carot ene int o ret inol in
chick int est inal mucosa wit h t he f ollowing procedures :
ARAT was assayed by measuring the formation of
3
labeled [ H] retinyl palmitate after incubating radioactive
3
50,000 cpm [ H] retinol and 10 nmol unlabeled retinol
with unlabeled palmitoyl-CoA in the presence of
microsomal ARAT.
The assay procedure were
basically the same as that described by Helgerud et al.,
(1982, 1983), and modified so that radioactive retinol
12
xviii
xix
C ha pte r III
Princ iple s o f
Spe c tro pho to m e te r
31
xx
Nut rient analysis in f eed or f ood is an import ant st ep in
composing a good diet t o provide all nut rient s needed f or
normal growt h and development . It is also import ant in
st udying or t he f at e of nut rient s during met abolism as has
been described in Chapt er I. Macro and micro nut rient s in f eed
or
f ood
(animal
product
is part
of
f ood
f or
human
consumpt ion), or in cert ain part s of body organs can be
det ermined qualit at ively or quant it at ively by various met hods.
Quant it at ive analyses can be done wit h t he aid of analyt ical
equipment called Spect rophot omet er. Spect rophot omet er is
an analyt ical inst rument which works on t he basis of simpl e
colourimet ric principles i. e. absorbance of light by colour
solut ion. Thus t his chapt er begins wit h an underst anding of
t he
light
and
colourimet er
principles.
Consequent ly,
spect rophot omet ry as t he science of spect rum of a compound
and as t he basis of quant it at ive det erminat ion of a nut rient
has a broad and growing applicat ion.
WHITE LIGHT / SUNLIGHT
Light t hat is emit t ed by t he sun and seen by human eyes as
whit e light consist s of various colours. If a beam of light
passes t hrough a prism, a range of colour light (purple, blue,
green, yellow, and red) will be emit t ed f rom t he prism (see
Figure 3. 1).
Light is an elect romagnet ic radiat ion which consist s of bands
of cert ain wavelengt hs and so does sunlight which
32
xxi
xxii
C ha pte r IV
De te rm ina tio n o f Lipid
In Se rum a nd Tissue
xxiii
61
To st udy t he ef f ect of f reshly made garlic powder on t he
serum concent rat ion of lipid in broilers, a publicat ion under
Mat erials & Met hods described t he f ollowing procedures :
One hundred male Ross 308 chicks were divided
into two equal groups. Group 1 received regular
broiler diet supplemented with 2% garlic powder.
Group 2 received regular broiler diet. All chickens
were slaughtered on day 50, every carcass was
weighed and its adipose tissue content was
determined. At the time of slaughtering, a 5-ml
blood sample was collected into a test tube from
each chicken. Tubes were labelled and then
centrifuged at 3,000 RPM for 10 min to collect blood
serum samples. The sera were analyzed to
measure total cholesterol and total triglyceride using
enzymatic colorimetric methods (Dehkordi et al.,
2009).
We can not e f rom t he above procedure t hat blood serum
samples were collect ed and t he concent rat ion of
t ot al
cholest erol and t ryglyceride were det ermined by colorimet ric
met hod or in anot her word by spect rophot omet ry t hat has
been discussed in Chapt er 2. The f irst st ep in det erminat ion of
cholest erol in serum is preparat ion of serum samples f rom
blood wit hdrawn f rom t he chicken or ot her experiment al
animals such as mouse, rat s, or ot her experiment al livest ock.
xxiv
62
C ha pte r V
De te rm ina tio n o f
Pro te in in Blo o d Se rum
87
xxv
To st udy t he act ivit y of t he enzyme involved in β-carot ene
met abolism in chicks f ollowing supplement at ion wit h nat ural
β-carot ene, a publicat ion under Mat erials & Met hods described
t he f ollowing procedures (Murwani, 2002):
Protein concentration in intestinal mucosa preparation
was measured following Lowry method using Bovine
Serum Albumin as standard. The same microsome
preparation was assayed for the enzyme activity which
catalysed the esterification of retinol (as the result of βcarotene splitting) into retinyl ester.
The above procedures described t he det erminat ion of prot ein
concent rat ion
of
int est inal
mucosa
preparat ion.
This
det erminat ion is needed t o express t he act ivit y of enzyme in
Unit per mg prot ein. Anot her mat erial and met hod can
describe t he det erminat ion of prot ein concent rat ion in serum
in order t o see normalit y or abnormalit y which might occur
f ollowing cert ain nut rient t reat ment .
Prot eins make up 6–8% of t he bl ood. The maj orit y of prot ein
f ound in serum consist of albumin, globulin, f ibrinogen and
prot hrombin. Normal prot ein in chicken serum is 3-3. 3 g/ dl,
and in human plasma is 7-7. 5 g/ dL. Prot ein in blood carry a
variet y of import ant f unct ions i. e. circulat ory, prot ect ion, and
regulat ion. As circulat ory f unct ion it t ransport s biomolecules
such as carbohydrat e, lipid, prot ein, vit amins, minerals,
hormones,
enzymes,
complement
component s,
prot ease
inhibit ors, et c. As regulat or, it regulat es pH, t emperat ure and
osmot ic balance. As
88
xxvi
xxvii
C ha pte r VI
De te rm ina tio n o f
Pho spho r in Fe e d o r
Fo o d
xxviii
95
A st udy was conduct ed t o evaluat e t he ef f icacy of 25hydroxycholecalcif erol
[ 25-(OH)D3]
to
minimize
t he
development of t ibial dyschondroplasia (TD) and improve
phyt at e phosphorus ret ent ion in Ross cockerels during t he
st art er
period
(Ledwaba and
Roberson,
2003).
Part
of
mat erials and met hods was described below.
A total of 240 1-d-old male broiler chicks were used for
each experiment. Ten chicks per pen were assigned
randomly to each of 24 pens in an electrically heated
battery brooder housed in a room without windows.
Room temperature was maintained at approximately
23°C . Feed and water were provided ad libitum in all
experiments.
The compositions of the corn-soybean
meal based diets are listed in Table 1.
Dietary 25-
(OH)D3 premix was donated and delivered from
commercial mill. Chromix oxide was used as an
external indicator at 0.10% of the basal diet to
determine
phytate
phosphorus
retention.
Feed
phosphorus was determined colorimetrically with a
spectrophotometer using the method described by
Gomori (1942).
In t he mat erial and met hod it is writ t en t hat det erminat ion of
phosphor in f eed was done by spect rophot omet ry.
Phosphor is a macro mineral essent ial f or bone f ormat ion.
Phosphor
combines
wit h
calcium
to
f orm
calcium
hydroxyapat it e which is t he const it uent of bone and t eet h.
96
xxix
xxx
Chapter VII
Determination of
Calcium
115
xxxi
A st udy was conduct ed t o evaluat e t he ef f icacy of 25hydroxycholecalcif erol
[ 25-(OH)D3]
to
minimize
t he
development of t ibial dyschondroplasia (TD) and improve
phyt at e phosphorus ret ent ion in Ross cockerels during t he
st art er period (Ledwaba and Roberson, 2003). Part of mat erials
and met hods is described below :
Blood samples
were obtained at 13 d of age via
cardiac puncture from one chick per pen and were
analyzed for each individual chick picked randomly as a
representative of the pen. The blood was centrifuged
for 15 min at 3,000 x g to extract serum and analyzed
for serum phosphorus (Goldenberg and Fernandez,
1966) and calcium (Moorhead and Biggs, 1974)
concentrations using the same spectrophotometer as
for feed phosphorus analysis.
Calcium is one of macro minerals which plays a cent ral role in
bone f ormat ion and maint anance in animals and human. In
chicken, 94% of egg shell is composed of calcium carbonat e.
When diet ary calcium is not suf f icient it can lead t o a
decrease in egg product ion, weight , and specif ic gravit y, f eed
consumpt ion, body weight , bone densit y and st rengt h. Diet ary
calcium t heref ore is essent ial f or f ast growing birds such as
broilers and egg producing birds such as layers. In layers,
t here is a high demand f or calcium during egg product ion.
When t his demand is not met it can lead t o t he occurence of
116
xxxii
xxxiii
C ha pte r VIII
De te rm ina tio n o f DNA
a nd RNA fro m C e lls o r
Tissue o f a n Anim a l
xxxiv
123
A st udy was conduct ed t o invest igat e t he development al
relat ionship bet ween f at t y acid composit ion in dif f erent lipid
f ract ions and st earoyl-CoA desat urase (SCD) gene expression in
st eer muscles during growt h. Under Mat erials & Met hods part
of t he procedures is described below :
Twenty male Korean Hanwoo steers were used in this
study. Steers were slaughtered at 6, 12, 18, 24 and 30months old, respectively. Muscle samples were taken
th
from longissimus dorsi between 5
vertebrae of steers.
th
and 6
lumbar
All samples were immediately
frozen in liquid nitrogen and stored at -80°C until
analysis.
Total RNA was isolated from loin muscle
using Trizol reagent (Life Technologies Inc., Grand
Island, NY, USA) according to the manufacturer’s
instructions. Following RNA isolation, the concentration
and purity of the prepared RNA were analyzed by
optical density measurements at 260/280 nm (Lee et
al., 2005).
The mat erials and met hods above showed t hat RNA was
det ermined by opt ical densit y measurement . Opt ical densit y
or abbreviat ed as OD is t he same as absorbance and indicat es
t hat it is measured spect rophot omet rically (See Chapt er 2).
DNA st ands f or Deoxyribo Nucleic Acid and RNA st ands f or Ribo
Nucleic Acid. DNA and RNA are t he genet ic codes t hat
det ermine t he charact erist ic of an individual. Nucleic acids
are f ound in t he nucleus of animals, plant s, and microbial
124
xxxv
xxxvi
C ha pte r IX
De te rm ina tio n o f
Enzym e Ac tivity
133
xxxvii
A st udy was conduct ed t o measure t he act ivit y of int est inal
phyt ase act ivit y in broiler chickens as af f ect ed by diet ary
calcium and vit amin D. Under mat erials and met hods t he
f ollowing procedures were described:
Hubbard x Peterson, male chicks were fed diets with or
without 0.21 mg/kg 25-OH D3 with varying dietary Ca
concentrations (4 or 9 g/kg) supplied primarily from
CaCO3 from 7 to 21 d of age (five pens/diet, three
birds/pen) in brooder battery cages. All birds were fed
diets calculated to contain 0.20% nonphytate P. Diets
contained Celite as an inert filler and as a digestive
marker at a minimum of 10 g/kg diet to allow for
determination of apparent phytate phosphorous (PP)
hydrolysis. Dietary Ca and total P were determined by
inductively
coupled
plasma-emission
spectroscopy
(ICP). Phytate-P content of diets was determined as
described below. At 7 d of age, birds were weighed
individually and allocated to experimental pens, such
that BW differences were minimized. At 21 d of age,
birds were euthanized by cervical dislocation.
The
duodenum and jejunum were rinsed in ice-cold saline,
and the mucosa was scraped and frozen (-80°C) for
future isolation of brush-border vesicles (BBV) and
subsequent
intestinal
phytase
determinations
(Applegate et al., 2003)
Enzymes are prot ein
t hat
speed
biochemical
react ions.
Enzymes have many applicat ions in t he f ield of animal and
134
xxxviii
xxxix
C ha pte r X
De te rm ina tio n o f To ta l
Phe no lic C o m po und
xl145
Polyphenols or phenolics are groups of subst ance nat urally
f ound in t he plant kingdom wit h chemical charact erist ics of
having aromat ic ring wit h one or more hydroxyl groups. They
are locat ed in t he vacuole and t end t o be wat er-soluble as
t hey are f ound in associat ion wit h sugars.
Included in
polyphernols are 1) simple phenols and t heir derivat ives such
as cat echol, eugenol, hydroxycinnamic acids (p-coumaric,
caf f eic,
f erulic
and
sinapic
acids),
and
coumarins,
2)
f lavonoids such as f lavonols and f lavones, chalcone and aurone
pigment s, f lavanones, and isof lavonoids, 3) ant hocyanins, 4)
Flavan-3-ols and f lavan-3, 4-diols, 5)Tannins which consist of
hydrolysable
t annins
and
condensed
t annins
(proant o-
cyanidins) (Scalbert and Williamson, 2000; Bennick, 2002).
Polyphenols can
phyt onut rient
excert
and
as
dual
ef f ect
i. e.
ant inut rient .
as f unct ional
As
f unct ional
phyt onut rient , many polyphenols have been shown t o have
ant ioxidant
propert ies which are import ant
syst em. The ant ioxidant propert ies of
in biological
isol at ed or pure
polyphenols are used t o enrich f eed and f ood t o improve
nut rit ional st at us of animals and human. Nat urally occurring
poliphenol in f eed mat erials such as sorghum can also be used
(Murwani, 2008a, b, c). The ant i-nut rient ef f ect of nat urally
occurring poliphenolic compounds of sorghum is well known. It
is det riment al t o poult ry when given in large amount s (Nyacot i
et al. , 1996). However, when it is used in small amount in t he
diet , it can exert it s biological f unct ion as immunomodulat or
146
xli
xlii
Re fe re nc e s
Alexander, R. R. , Grif f it hs, J. M. and Wilkinson, M. L. 1984. Basic
Biochemical Met hods. John Wiley & Sons Inc. New York.
AHMD. 2007. The American Herit age® Medical Dict ionary. Hought on
Mif f lin Company.
AOAC.
2000. Of f icial Met hods of Analysis. Associat ion of Of f icial
Analyt ical Chemist . Inc. , Vi rginia. USA.
AOAC. 1984. Of f icial Met hods of Analyses. Associat ion of Of f icial
Analyt ical Chemist s. Inc. , Arlingt on. USA.
Applegat e, T. J. , Angel , R. , and Classen, H. L. 2003. Ef f ect of
diet ary calci um, 25-hydroxycholecalcif erol , or bird st rain
on small int est inal phyt ase act ivit y in broiler chi ckens.
Poult ry Sci ence 82: 1140-1148.
Bennick, A. 2002. Int eract ion of plant polyphenols wit h salivary
prot eins. Crit ical Reviews in Oral Biology and Medicine.
13(2): 184-196
Bensadoun, A. and Rot hf iled, A. 1972. The f orm of absorpt ion of
lipids in t he chicken, Gal l us domest icus. Proceedi ngs of
t he Societ y f or Experiment al Biology and Medicine 41: 814817.
Bohac, C. E. , Rhee, K. S. , Cross, H. R. and Ono, K. 1984. Assessment
of met hodologies f or col orimet ric cholest erol assay of
meat . Journal Food Science 53(6): 1642-1693.
Coles, E. H. 1986. Vet erinary Cli nical Pat hology. 4t h Ed. W. B.
Saunders Company, Phi ladelphia.
Dehkordi, S. H. , Moghadam, A. Z. , Maghsoudi, N. , Aali, E. , Gerami,
R. , and Dehsadeghi, E. 2009. The ef f ect s of f resh garlic on
t he serum concent rat ion of t ot al cholest erol , t ot al
t riglyceride and adipose t issues of broi lers. Comparat i ve of
Clinical Pat hology. Published on line 25 Agust us 2009.
DiaSys. 2006. Technical Sheet of Triglyceride, Cholest erol and HDLCholest erol . DiaSys Diagnost ic Syst ems GmbH. Germany.
Dorey, R. C. , and Draves, J. 1998. Quant it at ive Anal ysis Laborat ory:
A New Approach.
Universit y of Cent ral Arkansas,
Depart ement of Chemist ry. Conwey. Arkansas.
157
xliii
xliv
APPENDIX A
Ato m ic We ig hts o f
The Ele m e nts
163
xlv
ATOMIC WEIGHTS OF THE ELEMENTS
Element
Symbol
Aluminum
Ant imony
Argon
Arsenic
Barium
Beryllium
Bismut h
Boron
Bromine
Cadmium
Calcium
Carbon
Cerium
Cesium
Chlorine
Chromium
Cobalt
Copper
Dysprosium
Erbium
Europium
Fluorine
Gadolinium
Gallium
Germanium
Gold
Haf nium
Helium
Holmium
Hydrogen
Indium
Iodine
Iridium
Iron
Krypt on
Lant hanum
Lead
Lit hium
Lut ecium
Magnesium
Manganese
Mercury
Molybdenum
Al
Sb
Ar
As
Ba
Be
Bi
B
Br
Cd
Ca
C
Ce
Cs
Cl
Cr
Co
Cu
Dy
Er
Eu
F
Gd
Ga
Ge
Au
Hf
He
Ho
H
In
I
Ir
Fe
Kr
La
Pb
Li
Lu
Mg
Mn
Hg
Mo
xlvi
164
At omic
weight
26. 98
121. 75
39. 95
74. 92
137. 34
9. 01
208. 98
10. 81
79. 90
112. 40
40. 08
12. 01
140. 12
132. 91
35. 45
52. 00
58. 93
63. 55
162. 50
167. 26
151. 96
19. 00
157. 25
69. 72
72. 59
196. 97
178. 49
4. 00
164. 93
1. 008
114. 82
126. 91
192. 22
55. 84
83. 80
138. 91
207. 21
6. 94
174. 97
24. 31
54. 94
200. 59
95. 94
Element
Symbol
Neodymium
Neon
Nickel
Niobium
Nit rogen
Osmium
Oxygen
Palladium
Phosphorus
Plat inum
Pot assium
Praseodymium
Prot act inium
Radium
Radon
Rhenium
Rhodium
Rubidium
Rut henium
Samarium
Scandium
Selenium
Silicon
Silver
Sodium
St rot ium
Sulf ur
Tant alum
Tellurium
Terbium
Thallium
Thorium
Thulium
Tin
Tit anium
Tungst en
Uranium
Vanadium
Xenon
Yt t erbium
Yt t rium
Zinc
Zirconium
Nd
Ne
Ni
Nb
N
Os
Ni
Nb
N
Os
O
Pd
P
Pt
K
Pr
Pa
Ra
Rn
Re
Rh
Rb
Ru
Sm
Sc
Se
Si
Ag
Na
Sr
S
Ta
Te
Tb
Ti
W
U
V
Xe
YB
Y
Zn
Zr
At omic
weight
144. 27
20. 18
58. 71
92. 91
14. 01
190. 2
16. 00
106. 4
30. 97
195. 09
39. 09
140. 91
231. 04
226. 03
222
186. 2
102. 91
85. 47
101. 07
150. 43
44. 96
78. 96
28. 09
107. 87
23. 00
87. 62
32. 06
180. 95
127. 60
158. 93
204. 39
232. 04
169. 93
118. 69
47. 90
183. 85
238. 03
50. 94
131. 30
173. 04
88. 91
65. 38
91. 22
APPENDIX B
Pe rio dic Ta b le
xlvii
165
166
xlviii
PERIODIC TABLE OF THE ELEMENTS
(IUPAC, 2008)
1
2
H
1.0079
3
III
13
II
2
4
5
IV
14
6
V
15
7
VI
16
8
9
Li
Be
B
C
N
O
6.941
11
9.0122
12
10.811
13
12.011
14
14.007
15
15.999
16
Na
Mg
3
4
22.990
19
24.305
20
K
Ca
Sc
Ti
39.098
37
40.078
38
44.956
39
47.88
40
Rb
Sr
85.468
87.62
55
56
21
Y
88.906
57-71
22
5
23
V
50.942
41
Zr
Nb
91.224
72
92.906
73
6
24
7
25
8
26
9
27
10
28
11
29
Cr
Mn
Fe
Co
Ni
Cu
51.996
42
54.938
43
55.847
44
58.933
45
58.69
46
63.546
47
Mo
95.94
74
Tc
{98}
75
12
30
Zn
65.39
48
Al
Si
26.982
31
28.086
32
Ga
69.723
49
Ge
72.61
50
P
30.974
33
As
74.922
51
S
32.066
34
Se
78.96
52
Ru
Rh
Pd
Ag
Cd
In
Sn
Sb
Te
101.07
76
102.91
77
105.42
78
107.87
79
112.41
80
114.82
81
118.71
82
121.75
83
127.60
84
Tl
204.38
Hf
Ta
W
Re
180.95
183.85
186.21
89-103
104
105
106
Fr
Ra
**Ac
Unq
Unp
Unh
At om i c w ei ght s ar e based on 12C = 12 and confor m t o t he 1987 IUPAC r epor t val ues r ounded t o 5
si gni fi cant di gi t s. Num ber s i n { } i ndi cat e t he m ost st abl e i sot ope.
226.03
227.03
{261}
{262}
{263}
57
58
59
60
64
65
66
67
207.2
68
Bi
208.98
69
Po
I
126.90
85
178.49
63
Pb
Kr
83.80
54
*La
** Actinides
Hg
200.59
Br
79.904
53
138.91
62
Au
196.97
Ar
39.948
36
Ba
61
Pt
195.08
{209}
70
Ne
20.180
18
Cl
137.33
88
* Lanthanides
Ir
He
4.0026
10
35.453
35
132.91
87
190.2
192.22
F
18.998
17
Cs
{223}
Os
VII
17
At
{210}
71
*La
Ce
Pr
Nd
Pm
Sm
Eu
Gd
Tb
Dy
Ho
Er
Tm
Yb
Lu
138.91
140.12
140.91
144.24
{145}
150.36
151.97
157.25
158.93
162.50
164.93
167.26
168.93
173.04
174.97
89
90
91
92
93
94
97
98
99
100
101
102
103
95
96
**Ac
Th
Pa
U
Np
Pu
Am
Cm
Bk
Cf
Es
Fm
Md
No
Lr
227.03
232.04
231.04
238.03
237.05
{244}
{243}
{247}
{247}
{251}
{252}
{257}
{258}
{259}
{262}
Xe
131.29
86
Rn
{222}
G LO SSARY
AAS
:
At omic Aborpt ion Spect rophot omet er,
an analyt ical inst rument which ionizes
mineral sample and t he absorpt ion of
t he
ionized
at om
is
measured
spect rophot omet rically.
Abdominal fat
:
Fat which is deposit ed in t he abdominal
cavit y of broilers or commercial meat
chicken.
Albumin
:
A t ype of prot ein t hat is soluble in
wat er and in wat er half sat urat ed wit h
a salt such as ammonium sulf at e. Serum
albumin is a component of blood serum.
Egg and milk also cont ain albumin.
Seeds cont ain very small amount s of
albumins.
Antibody
:
Prot ein which is secret ed by specialized
immune cells i. e. B cells. It prot ect s
t he body against f oreign ant igen.
Antibody titers
:
The quant it y of ant ibody produced by B
cells in response t o ant igen.
Antigen
:
Spesif ic
molecules
of
f oreign
microorganism or cells or t issue.
Aqueous phase
:
liquids t hat are wat er-soluble. Because
t hey mix wit h wat er, t hey f orm a liquid
phase which are not separable. For
example, met hanol is mixed wit h
wat er, t heref ore t hey do not f orm a
separat e phase.
Arachidonic
acid
:
is a carboxylic acid. Arachidonic acid is
import ant because t he human body
uses it as a st art ing mat erial in t he
167
xlix
l
Inde x
A
AAS, 9, 117, 118, 167
Abdominal f at , 69, 136, 139, 167
absorbance, 32, 37-41, 43, 45-47, 49, 50, 56, 58, 59, 73, 74,
76, 79, 81, 84, 91-93, 106, 107, 109, 120, 121, 124, 132,
137, 138, 141, 143, 149-151, 154, 155, 168.
acet ic acid, 25, 27-29, 64, 81, 91, 110, 118, 136
acid, 5-7, 13, 19-30, 64, 65, 67, 68, 81, 91, 96, 98, 99, 110,
118, 124, 125, 132, 136, 137, 141, 146-152, 154
acidit y, 21, 25
ad l i bi t um , 12, 96
addit ives, 129
Adenosine Tri Phosphat e (ATP), 7
adipocyt es, 68
Adipose, 62, 68
ADP, 96
agarose, 5
alanine, 140-142
ALAT, 140-142
albumen, 82
alkaline, 89, 131
alkalinit y, 21, 25
amino acid, 5, 28, 141
aminohexanoic acid, 13, 19
Amino-Napht ol-Sulf onic Acid, 98, 99, 110
ammonia, 25, 26, 135
ammonium molybdat e, 107, 112
ammonium sulf at e, 155, 167
ammonium vanadat e, 98, 107, 137
ampere, 13
animal welf are, 3
ant ibiot ic, 17, 138, 142-144, 160
ant ibody, 5, 89, 147
ant icoagulant , 64
173
li
lii
liii