TEC HNIQ UES O F ANALYSES IN
NUTRITIO NAL BIO C HEMISTRY
Retno Murwani
Head of Labor at or y of Nut r i t i onal Bi ochemi st r y Dept . of Nut r i t i on and Feed Sci ence
Facul t y of Ani mal Sci ence Di ponegor o Uni ver si t y Semar ang
v
Book : Techniques of Analyses in Nut rit ional Biochemist ry; Ret no Murwani (aut hor); 1st Ed; Badan Penerbit Undi p Semar ang; 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 copyright2009
Al l r i ght s r eser ved
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
vii 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
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 of an Animal ... 123 IX Det erminat ion of Enzyme Act ivit y... 133
X Det erminat ion of Tot al Phenolic Compound... 145
Ref erences ... 157
ix Appendix B
Periodic Table... 165
Glossary ... 167
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
2. 4. 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
4. 2. An example of calculat ion in t he det erminat ion of HDL cholest erol in serum sample ... 76
6. 1. Dilut ion of P st ock solut ion t o obt ain a series of P st andard concent rat ion ... 104
6. 2. 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
xi of colour (colour band) which consist s of purple, blue, green, yellow, and red (Whit e et al . , 1965; absorbance and concent rat ion ... 41
3. 5. 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
3. 6. Out line of a spect rophot omet er (modif ied f rom Segel, 1975) ... 43
3. 8. Spect ronic 20 (DIGITAL Spect rophot omet er f rom Thermo Fisher Scient if ic, 2009) ... 45
3. 9. Spect ro UVS-2700 is a double-beam UV-VIS Spect rophot omet er wit h eight cuvet t e holders (Labomed, 2001) ...
3. 12. UV-1201 Spect rophot omet er rear view (Shimadzu,
1994) ... 51
3. 13. UV-1201 Spect rophot omet er rear view (Shimadzu, 1994) ... 51
3. 14. 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 (modified from DiaSys, 2006; Murwani and Bayuardhi, 2007)...
72
4. 4. 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 ...
73
4. 5. Procedures f or det erminat ion of HDLcholest erol (modified from DiaSys, 2006; Murwani, 2008b) ... 77
xiii
(modified from DiaSys, 2006; Murwani, 2008b) ... 79
4. 7. Fresh sample meat is weighed ... 83 reagent and developed int o color solut ion Concent rat ion of st andard solut ion f rom lef t t o ready f or absorbance reading (sorghum sample) ...
C ha p te r I
Intro d uc tio n to
xv 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
xvii
C ha p te r II
Units o f a Nutrie nt
C o nc e ntra tio n
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 er ials & 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 β -carotene/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 :
C ha p te r III
Princ ip le s o f
xxi 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 var ious 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
xxiii
C ha p te r IV
De te rm ina tio n o f Lip id
In Se rum a nd Tissue
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
xxv
C ha p te r V
De te rm ina tio n o f
Pro te in in Blo o d Se rum
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
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
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 β -carotene 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
C ha p te r VI
De te rm ina tio n o f
Pho sp ho r in Fe e d o r
xxix A st udy was conduct ed t o evaluat e t he ef f icacy of
25-hydroxycholecalcif erol [ 25-(OH)D3] t o 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
Phosphor is a macro mineral essent ial f or bone f ormat ion.
Phosphor combines wit h calcium t o f orm calcium
hydroxyapat it e which is t he const it uent of bone and t eet h. 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.
xxxi
Chapter VII
Determination of
Calc
ium
A st udy was conduct ed t o evaluat e t he ef f icacy of
25-hydroxycholecalcif erol [ 25-(OH)D3] t o 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
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 r engt 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 as
C ha p te r VIII
De te rm ina tio n o f DNA
a nd RNA fro m C e lls o r
xxxv 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
30-months old, respectively. Muscle samples were taken
from longissimus dorsi between 5th and 6th lumbar
vertebrae of steers. 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
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 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).
xxxvii
C ha p te r IX
De te rm ina tio n o f
Enzym e Ac tivity
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
Enzymes are prot ein t hat speed biochemical react ions.
C ha p te r X
De te rm ina tio n o f To ta l
Phe no lic C o m p o und
xli 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) simpl e 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 excert dual ef f ect i. e. as f unct ional
phyt onut rient and as ant inut rient . As f unct ional
phyt onut rient , many polyphenols have been shown t o have
ant ioxidant propert ies which are import ant in biological
syst em. The ant ioxidant propert ies of 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
xliii
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 Yor k. AHMD. 2007. The American Herit age® Medical Dict ionary. Hought on
Mif f lin Company. 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. t he Societ y f or Experiment al Biology and Medicine 41: 814-817.
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 er inary Cli nical Pat hology. 4t h Ed. W. B. Saunders Company, Phi ladelphia.
Dehkor di, 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 ser um 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
HDL-Cholest 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.
xlv
APPENDIX A
Ato m ic We ig hts o f
The Ele m e nts
xlvii
APPENDIX B
Pe rio d ic Ta b le
xl
xlix
G LO SSA RY
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 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
li aminohexanoic acid, 13, 19
