Echotexturanalyse des Lebergewebes zur nicht-invasiven Bestimmung des Leberfettgehaltes bei Milchkühen
INAUGURAL - DISSERTATION
zur Erlangung des Grades eines Doktors der Veterinärmedizin
- Doctor medicinae veterinariae - ( Dr. med. vet. )
vorgelegt von A l o i s H a u d u m aus Haslach an der Mühl
Österreich
Hannover 2009
Wissenschaftliche Betreuung: Univ. Prof. Dr. Jürgen Rehage
Klinik für Rinder
1. Gutachter: Univ. Prof. Dr. Jürgen Rehage 2. Gutachter: Univ. Prof. Dr. Peter Stadler
Tag der mündlichen Prüfung: 17. November 2009
Gefördert von der
Meiner Familie,
insbesondere meinen Eltern, in Liebe und Dankbarkeit sowie meinem Opa zum Andenken
1. EINLEITENDE LITERATURÜBERSICHT 11 1.1. Prävalenz und wirtschaftliche Bedeutung der Leberverfettung
beim Milchrind
11
1.2. Entstehung der Leberverfettung 12
1.3. Histologische und makroskopische Veränderungen bei Fetteinlagerung in die Leber
13
1.4. Dynamik der Leberverfettung 14
1.5. Bestimmung des Leberfettgehaltes 15
1.6. Risiken der Leberbioptatentnahme 17
1.7. Nicht-invasive Verfahren zur Bestimmung des Leberfettgehaltes 17 1.8. Fragestellungen und Zielsetzungen dieser Arbeit 20
2. KAPITEL 1 21
ANALYSIS OF TOTAL LIPID AND TRIACYLGLYCEROL CON- TENT IN VERY SMALL LIVER BIOPSY SAMPLES IN CATTLE
Introduction 25
Material and Methods 26
Results 31
Discussion 34
Conclusion 39
3. KAPITEL 2 59
COMPUTER-AIDED B-MODE ULTRASOUND DIAGNOSIS OF HEPATIC STEATOSIS: A FEASIBILITY STUDY
Introduction 61
Material 62
Methods 63
Results 68
Discussion 69
DAIRY COWS WITH CALIBRATED ULTRASONOGRAPHIC IMAGE ANALYSIS
Introduction 79
Material and Methods 82
Results and Discussion 89
Conclusion 96
5. ÜBERGREIFENDE DISKUSSION 115
6. ZUSAMMENFASSUNG 120
7. SUMMARY 123
8. LITERATURVERZEICHNIS 127
Literaturübersicht und Übergreifende Diskussion
Acetyl-CoA Acetyl-Coenzym A B-Mode brightness mode bzw. beziehungsweise
ca. circa
CAUS Computer-Aided Ultrasound Diagnosis et al. et alii
Fig. Figure
FW Leberfrischgewicht
g Gramm
ggr. geringgradig
h Stunden
hgr. hochgradig
HPLC high pressur liquid chromatography
max. maximal
mg Milligramm
mgr. mittelgradig
NEFA non-esterified fatty acids r Korrelationskoeffizient
r² Bestimmtheitsmaß
USA United States of America US$ United States Dollar
TAG Triacylglycerol
TL Totallipid
u. und
u. a. unter anderem
VLDL very low density lipoproteins
vs. versus
z. B. zum Beispiel
BP break-point
FW wet fresh weight
Lubrol nonaethylene glycol monododecyl ether RSD residual standard deviation
TAG triacylglycerol
TL total lipid
Kapitel 2
AGC automatic gain compensation CAUS Computer-Aided Ultrasound Diagnosis FWHM full-width-at-half-maximum
LGC lateral gain correction
LUT look-up-table
MOD magneto optical disk
ROI region-of-interest
TGC time gain compensation
AGC Automatic Gain Control
AUC Area-under-the-Curve
AxSp axial speckle size B brightness
CAUS Computer-Aided Ultrasound Diagnosis
dB decibel
FW liver fresh weight grl gray level
LatSp lateral speckle size
LDA left-sided abomasal displacement
LUT Look-up Table
Mean/SD mean over the standard deviation MeanEcho mean echo level
ResAtt residual attenuation
ROC Receiver Operating Characteristic
ROI Region-of-Interest
TAG triacylglycerol
TAGmeas measured triacylglycerol TAGpred predicted triacylglycerol
US ultrasonographic
1. EINLEITENDE LITERATURÜBERSICHT
1.1. Prävalenz und wirtschaftliche Bedeutung der Leberverfettung beim Milch- rind
Prävalenz
Die Leberverfettung ist bei hochleistenden Milchkühen die wichtigste und häufigste Lebererkrankung und tritt vornehmlich in den ersten Wochen post partum auf (MOR- ROW 1976; ROBERTS et al. 1981; BOBE et al. 2004). Etwa 40 bis 60% der Tiere können von einer mgr. bis hgr. Leberverfettung betroffen sein (REID 1980;
GERLOFF et al. 1984, 1986; JORRITSMA et al. 2001; RAOOFI et al. 2001). Dies entspricht auch dem typischen Verteilungsmuster der Leberfettgehaltsgrade von je einem Drittel nicht bis ggr., mgr. bzw. hgr. bei Kühen mit einer Labmagenverlagerung nach links (HOLTENIUS u. NISKANEN 1985; MUYLLE et al. 1990; REHAGE et al.
1996). Labmagenverlagerungen betreffen etwa 3 bis 5% der frühlaktierenden Milch- kühe (CONSTABLE et al. 1992; GEISHAUSER 1995; KELTON et al. 1998;
LEBLANC et al. 2005). In späteren Laktationsstadien werden nur bei etwa 20% der Kühe Leberverfettungen unterschiedlichen Grades beobachtet (RAOOFI et al. 2001;
NAGARAJAN et al. 2007).
Wirtschaftliche Bedeutung
Ökonomische Bedeutung gewinnt die Leberverfettung durch die Reduktion von Milchleistung und Fruchtbarkeit sowie das erhöhte Risiko des Auftretens anderer peripartaler Erkrankungen, wie Labmagenverlagerungen oder Mastitiden (MORROW 1976; GERLOFF et al. 1986; HERDT 1988; REHAGE et al. 1996; WENSING et al.
1997; JORRITSMA et al. 2000; BOBE et al. 2004; GEELEN u. WENSING 2006;
MULLIGAN u. DOHERTY 2008). Auch entwickeln Kühe mit zunehmendem Leber- fettgehalt eher eine Leberinsuffizienz (bis 5,3 faches Risiko; REHAGE et al. 1996).
Die Leberverfettung zieht infolgedessen als Ursache und Wegbereiter anderer Er- krankungen tierärztliche Behandlungskosten, verlängerte Zwischenkalbezeiten, Milchleistungsrückgang und die vorzeitige Verwertung betroffener Tiere nach sich (BOBE et al. 2004). Die direkt verursachten Kosten sind schwierig abzuschätzen.
Der Grad der Leberverfettung und die Anzahl der betroffenen Tiere sind nur über die Analyse eines Leberbioptats am Einzeltier genau zu erfassen (BOBE et al. 2004). Da dies in der Praxis nicht routinemäßig erfolgt, lassen sich durch Folgeerkrankungen verursachte Kosten nicht in jedem Fall zuverlässig auf die Leberverfettung beziehen.
Die durchschnittlichen Kosten beispielsweise einer Ketose, eine Stoffwechselerkran- kung, welche pathogenetisch sehr eng mit der Leberverfettung verbunden ist, (VEENHUIZEN et al. 1991) werden auf 145 US$ pro Fall geschätzt. Bei einem Be- stand von ca. neun Millionen Milchkühen in den USA belaufen sich die Kosten nur allein für die mit einer Leberverfettung vergesellschafteten Ketosen bei einer Keto- seprävalenz von 4,8% auf über 60 Millionen US$ jährlich (BOBE et al. 2004). Umge- rechnet auf Deutschland, wo knapp fünf Millionen Milchkühe gehalten werden, wür- den sich die Kosten demnach allein für die durch die Leberverfettung hervorgerufene Ketose auf jährlich ca. 35 Millionen US$ belaufen.
1.2. Entstehung der Leberverfettung
Ursächlich kann die Leberverfettung auf eine negative Energiebilanz infolge einer Imbalanz zwischen nutritiver Energieaufnahme und Energieabgabe über die Milch- produktion zurückgeführt werden (COLLINS u. REID 1980; HERDT 1988). Dieses Defizit ruft eine hormonell induzierte Mobilisierung von körpereigenen Energiereser- ven, insbesondere eine Lipolyse hervor (BELL 1995). Dabei werden in den Fettde- pots verstärkt NEFA aus TAG freigesetzt (HERDT 2000). Reguliert wird dies im We- sentlichen durch die Hormone Insulin, Glucagon, Epinephrin, Norepinephrin und das bovine Wachstumshormon (HERDT 2000).
Insulin, als metabolisches Schlüsselhormon, senkt den Blutglucosespiegel, indem es den Glucoseverbrauch im Muskelgewebe steigert und gleichzeitig die hepatische Gluconeogeneserate reduziert. Im Fettgewebe stimuliert Insulin die Lipogenese und hemmt die Lipolyse. Damit unterdrückt es die Mobilisation der NEFA. Wenn infolge der negativen Energiebilanz die Blutkonzentration der Glucose fällt, sinkt die Insulin- freisetzung aus den -Zellen des Pankreas (LÖFFLER u. PETRIDES 1997), was zu
Gluconeogenese. Bei niedrigem Blutglucosespiegel steigt die Glucagonkonzentration im Blut. Weiterhin wichtig zur Kontrolle und Regulation des Fettgewebs- Metabolismus sind die Catecholamine, Epinephrin und Norepinephrin. Sie stimulie- ren die Lipolyse und werden im Falle einer negativen Energiebilanz verstärkt ausge- schüttet. Auch das bovine Wachstumshormon führt zu einer verringerten Lipogenese und stimuliert die Lipolyse aus den Fettgewebsspeichern (HERDT 2000).
Die Plasmakonzentrationen der aus peripheren Fettdepots freigesetzten NEFA korre- lieren eng mit dem Grad der Fettmobilisierung und der hepatischen NEFA-Aufnahme (etwa 40% des Gesamtangebots; DUNSHEA et al. 1989). In der Leber können NEFA in den Citratzyklus eingeschleust, zu Ketonkörper metabolisiert oder zu TAG reve- restert werden. Für die ersten beiden Stoffwechselwege werden die NEFA über den Carnithin-Shuttle in die Mitochondrien der Hepatozyten transportiert. Dort werden sie ß-oxidativ zu Acetyl-CoA gespalten und können als C2-Körper über Ankopplung an Oxalacetat in den Citratzyklus eingeschleust werden (KREBS 1961, 1966; BAIRD 1982; LOMAX 1992). Da aber die Gluconeogenese Vorrang genießt (WEEKS 1991), steht dafür nicht genug Oxalacetat als Substrat zur Verfügung. Daher wird ein erheb- licher Anteil des Acetyl-CoA zu Ketonkörpern (Aceton, Acetacetat, Beta- Hydroxybutyrat) verstoffwechselt (KREBS 1966). Ein weiterer Teil der NEFA wird bereits im Cytosol zu TAG reverestert (LOMAX 1992). Unter physiologischen Bedin- gungen werden die TAG an Lipoproteine (hauptsächlich VLDL) gebunden und aus dem Cytosol ausgeschleust. Aus bisher ungeklärter Ursache ist die Syntheserate der Lipoproteine bei Milchkühen in der Hochlaktation jedoch vermindert, so dass die TAG in der Leber akkumulieren (KLEPPE et al. 1988; MAZUR et al. 1988; PULLEN et al.
1989; DRACKLEY 1999; HERDT 2000). TAG hat als Speicherform des Fettes somit eine besondere Bedeutung für die Leberverfettung (GRUMMER 1993; DRACKLEY 1999).
1.3. Histologische und makroskopische Veränderungen bei Fetteinlagerung in die Leber
Die TAG werden in der Leberzelle in Form von Fetttröpfchen gespeichert (DRACKLEY 1999), was zu einer Volumenzunahme der Hepatozyten führt (REID u.
COLLINS 1980; JOHANNSEN et al. 1993). In Abhängigkeit vom Grad der Fetteinla- gerung handelt es sich initial um eine kleintropfige, im Folgenden eher um eine großtropfige Verfettung (CEBRA et al. 1997; KALAITZAKIS et al. 2007). Innerhalb des Leberläppchens beginnt die Leberverfettung um die Zentralvene (zentrolobulär) und breitet sich zunehmend über intermediär und peripher bis panlobulär aus (HERDT et al. 1982; MERTENS 1992; CEBRA et al. 1997; KALAITZAKIS et al.
2007). Die Fetteinlagerung erfolgt weitgehend homogen über das gesamte Organ (GRÖHN u. LINDBERG 1982; GAAL u. HUSVETH 1983). Im fortgeschrittenen Sta- dium führt die Hypertrophie der Hepatozyten zu einer auch makroskopisch nach- weisbaren Parenchymschwellung der Leber. Da die Organkapsel straff gespannt bleibt, stumpfen infolgedessen die Leberränder ab (MORROW et al. 1979; HERDT 1988).
1.4. Dynamik der Leberverfettung
Im Zuge der fortschreitenden Fetteinlagerung steigt im Lebergewebe neben dem TAG wenn auch weniger deutlich der Gehalt weiterer Lipide z. B. Cholesterol, Cho- lesterolester und NEFA. Die Konzentration an Phospholipiden bleibt dagegen nahezu unverändert (BRUMBY et al. 1975; REID et al. 1977; COLLINS u. REID 1980; BOBE et al. 2004). Insgesamt gesehen, nimmt der proportionale Anteil der TAG am TL Ge- halt der Leber zu (GAAL et al. 1983; REID u. ROBERTS 1983; HERDT 1988;
STAUFENBIEL et al. 1993). In vielen Studien werden beide Parameter gemessen (REID et al. 1977; GAAL et al. 1983; DRACKLEY et al. 1991, 1992; STAUFENBIEL et al. 1992, 1993; PERSHING et al. 2002; SELBERG et al. 2005; CARLSON et al.
2006; KALAITZAKIS et al. 2006; CARLSON et al. 2007; KALAITZAKIS et al. 2007;
STAUFENBIEL et al. 2007; MCFADDEN et al. 2008). Auffällig ist dabei, dass insbe- sondere bei hohen TL Gehalten die prozentualen Anteile der TAG an den TL erheb- lich variieren. Während einige Autoren bei hepatischen TL Gehalten ab ca. 100 mg/g FW TAG Anteile von etwa 20 bis 30% finden (KALAITZAKIS et al. 2006, 2007), be- richten andere Autoren von 60 bis 70% (STAUFENBIEL et al. 1992; SELBERG et al.
keine detaillierten Angaben zur analytischen Prozedur oder zur Präzision der Metho- de gemacht. Es kann daher nicht endgültig ausgeschlossen werden, dass vor allem mangelnde Präzision der Analytik Ursache der unterschiedlichen prozentualen TAG Anteile ist. Vorstellbar wäre, dass beispielsweise bei Verwendung enzymatischer Se- rum-Testkits bei hohen Leberfettgehalten die Bestimmung von TAG unvollständig bleibt.
1.5. Bestimmung des Leberfettgehaltes Bioptat
Zur Quantifizierung des Leberfettgehaltes im Rahmen von wissenschaftlichen Stu- dien (MORROW 1976; HERDT 1988; BOBE et al. 2004) oder auch zu diagnosti- schen Zwecken im Rahmen der Herdenbetreuung (GELFERT et al. 2004) ist bislang die Untersuchung von Leberbioptaten unentbehrlich. Aufgrund der weitgehend ho- mogenen Fettverteilung im Lebergewebe genügen verhältnismäßig kleine Bioptat- mengen, um den Verfettungsgrad des Gesamtorgans widerzuspiegeln (GRÖHN u.
LINDBERG 1982; GAAL u. HUSVETH 1983). So gilt die Entnahme eines Bioptates als ausreichend, um den Grad der Organverfettung festzustellen (REID 1980;
HERDT 1988; WEST 1990). Die Leberbioptate können entweder mittels Biop- sienadeln (GRÖHN u. LINDBERG 1982; SIMPSON 1985; BUCKLEY et al. 1986;
SWANSON et al. 2000) oder Feinnadelschneidbiopsiegeräten (HERDT et al. 1983;
HERDT 1988; SCHOLZ et al. 1989) gewonnen werden.
Analytik
Die Analytik des Leberfettgehaltes der Bioptate kann histologisch (COLLINS u. REID 1980; COLLINS et al. 1985; JOHANNSEN et al. 1988; MERTENS 1992), über die Flotationsmethode (HERDT et al. 1983) oder mittels biochemischer Verfahren (HARA u. RADIN 1978; BICKHARDT et al. 1988; VEENHUIZEN et al. 1991) erfol- gen. Bei den ersten beiden Varianten wird der Verfettungsgrad indirekt geschätzt.
Der Vorteil der histologischen Untersuchung besteht darin, dass neben dem Verfet- tungsgrad sowohl die Größe der Fetttropfen und deren Verteilung innerhalb der Le- berzelle als auch pathomorphologische Veränderungen direkt beurteilt werden kön-
nen (COLLINS u. REID 1980; GAAL et al. 1983). Die Abschätzung des Lipidgehaltes über die Änderung des spezifischen Gewichtes des Lebergewebes (Flotations- methode) ist ein für Praxisbedingungen geeigneter Schnelltest, der auf Kupfersulfat- lösungen unterschiedlicher Dichte basiert (HERDT et al. 1983). Der Vorteil der bio- chemischen Verfahren besteht darin, dass TL und TAG direkt bestimmt werden kön- nen. TL wird üblicherweise mittels organischer Lösungsmittel extrahiert und an- schließend gravimetrisch erfasst (FOLCH et al. 1957; ATKINSON et al. 1972; HARA u. RADIN 1978). Für dieses Verfahren werden Gewebemengen von bis zu 1000 mg verwendet (HARA u. RADIN 1978; GAAL et al. 1983; KALAITZAKIS et al. 2007).
TAG als Speicherform der Lipide wird dabei zunächst auf enzymatischem (BUCOLO u. DAVID 1973; ZIEGENHORN 1975; BICKHARDT et al. 1988) oder biochemischem (CHIN et al. 1971; SOLONI 1971) Weg hydrolytisch gespalten. Danach erfolgt der Nachweis indirekt kolorimetrisch (FLETCHER 1968), fluorometrisch (NEMETH et al.
1986) oder in Verbindung mit einer high performance liquid chromatography (HPLC;
KRUEMPELMAN u. DANIELSON 1982). Hierfür werden Gewebemengen von bis zu 10000 mg (BULTER et al. 1961; BICKHARDT et al. 1988) eingesetzt. Die Analyse von TAG erfolgt beim Rind üblicherweise unter Verwendung kommerzieller Serum- Testkits (GAAL et al. 1983; DRACKLEY et al. 1991, 1992). TAG werden dabei mittels Lipase hydrolytisch gespalten und das Glycerol enzymatisch erfasst (FLETCHER 1968; FOSTER u. DUNN 1973; DRACKLEY et al. 1992). Seltener wird zur TAG Be- stimmung eine Dünnschichtchromatographie verwendet (REID et al. 1977).
Neben erhöhtem Arbeits- und Zeitaufwand für Gewinnung und Aufarbeitung großer Leberbioptate ist es auch aus Gründen des Tierschutzes erstrebenswert, die Biop- tatmenge zu reduzieren. Um dies zu erreichen und gleichzeitig den Aufwand für die getrennte Analyse von TL und TAG zu verringern, empfiehlt es sich, beide Parameter aus einem Ansatz zu bestimmen. VEENHUIZEN et al. (1991), DRACKLEY et al.
(1991) und BOBE et al. (2008) analysierten so TL und TAG aus Lebergewebeproben (ca. 1000 – 3000 mg) vom Rind. Die Autoren machten allerdings keine Angaben zu benötigten Gewebemengen, zum detaillierten Vorgehen und über Genauigkeit und
1.6. Risiken der Leberbioptatentnahme
Obwohl es sich bei der Feinnadelschneidbiopsie (SCHOLZ et al. 1989), welche pro Entnahme nur ca. 15 bis 30 mg Gewebe (HERDT 1988) liefert, um ein minimal- invasives Verfahren zur Bioptatentnahme handelt, besteht eine geringe aber stetige Gefahr von Infektionen und Blutungen (SMART u. NORTHCOTE 1985; SMITH et al.
1997; SWANSON et al. 2000). Aus humanmedizinischen Untersuchungen geht her- vor, dass es sich trotz Lokalanästhesie bei der Bioptatentnahme um einen schmerz- haften Prozess handelt, welcher auch 24 h später noch Schmerzen bereitet (EISENBERG et al. 2003). Darüber hinaus ist die Gewinnung und Analyse eines Le- berbioptates mit hohem Arbeits- und Zeitaufwand verbunden, weshalb zwischen Bi- optatentnahme und dem Vorliegen des Ergebnisses in jedem Fall eine gewisse Zeit- spanne liegt. Seit längerem gibt es daher Bestrebungen den Grad der Leberverfet- tung nicht-invasiv zu bestimmen.
Versuche den Grad der Leberverfettung bei Milchkühen beispielsweise allein über Blut- (STAUFENBIEL et al. 1993; CEBRA et al. 1997) oder Milchuntersuchungen (STAUFENBIEL et al. 1993) sowie über Body Condition Scoring (REID 1983;
STAUFENBIEL et al. 1993) oder Rückenfettdickemessungen (SCHRÖDER u.
STAUFENBIEL 2006) abzuschätzen, lieferten keine zuverlässigen Ergebnisse.
1.7. Nicht-invasive Verfahren zur Bestimmung des Leberfettgehaltes Ultraschall
Eine in der Human- als auch Veterinärmedizin bewährte, nicht-invasive Methode zur Gewebeanalyse innerer Organe ist die Sonographie. Sie wurde bereits zur Bestim- mung des Leberfettgehaltes beim Rind getestet (GROTE 1992; LAUENER 1993;
ACORDA et al. 1994; DELLING 2000; BOBE et al. 2008). Grundlage des Einsatzes in der Fettleberdiagnostik sind zwei Hypothesen. Zum einen geht, wie bereits be- schrieben, der Anstieg des Leberfettgehaltes mit einer Volumenzunahme des Organs einher (MORROW et al. 1979), zum anderen führt er zu einer Änderung der Echotex- tur des Gewebes (BRAUN 1990; GROTE 1992; LAUENER 1993; ACORDA et al.
1994; BRAUN u. GERBER 1994; DELLING 2000).
Da die Leberdimensionen individuell schwanken und die Schwellung des Organs auch andere Ursachen (Abszesse, Tumore, Blutstauung) haben kann (BRAUN et al.
1996), waren bisherige Versuche, den Leberfettgehalt über die sonographische Ver- messung der Volumenzunahme des Organs zu erfassen, nicht erfolgreich (GROTE 1992; LAUENER 1993; DELLING 2000).
Die durch Fetteinlagerung bedingte Änderung der Echotextur des Lebergewebes wurde bisher sowohl subjektiv (GROTE 1992; LAUENER 1993; DELLING 2000) als auch digital (ACORDA et al. 1994; BOBE et al. 2008) erfasst. Subjektive Erfahrungen zeigten einheitlich, dass die Leber mit steigendem Fettgehalt in ihrer Echogenität (Bildgrauwerte) zunimmt und im Ultraschallbild heller erscheint (GROTE 1992;
LAUENER 1993; ACORDA et al. 1994; DELLING 2000). Über andere transkutan er- hobene Texturparameter gibt es unterschiedliche Auffassungen. So nehmen offenbar Echodichte und Grobkörnigkeit des Echomusters (GROTE 1992; LAUENER 1993) sowie Echoabschwächung bei vermehrter Fetteinlagerung zu (DELLING 2000), wo- bei letzteres nicht in jedem Fall bestätigt wurde (LAUENER 1993). Auch scheint es Unterschiede zwischen transkutan und intraoperativ erhobenen Ultraschallbildern zu geben. DELLING (2000) stellte transkutan nur bei der Echodichte, intraoperativ hin- gegen sowohl bei der Echodichte als auch bei der Grobkörnigkeit der Echos eine Zunahme mit steigendem Fettgehalt fest. Übereinstimmend schlussfolgerten alle Au- toren, dass es anhand subjektiv erhobener Echotexturparameter nur bedingt möglich war, den Leberfettgehalt vorherzusagen (GROTE 1992; LAUENER 1993; ACORDA et al. 1994; DELLING 2000). Hinzu kommt, dass die subjektive Beurteilung von Le- berultraschallbildern hohe inter- und intraindividuelle Unterschiede hinsichtlich des Untersuchers aufweist und dadurch eine geringe Genauigkeit und Vergleichbarkeit der Ergebnisse erreicht wird (STRAUSS et al. 2007). Zur Vermeidung dieser Subjek- tivität wurde die digitale quantitative Analyse von Ultraschallbildern in der Humanme- dizin bereits mehrfach getestet. Grundsätzlich gibt es dafür zwei Ansätze. Einer ba- siert auf der Spektralanalyse von Radiofrequenz-Rohdaten der Echogramme (THIJSSEN 2000; LIZZI et al. 2006). Ein zweiter Ansatz beruht auf der quantitativen
al. 1987). Dies ermöglicht somit potentiell eine weitere, digitale quantitative Auswer- tung konventioneller B-Mode Ultraschallbilder.
Veterinärmedizinische Untersuchungen zur nicht-invasiven Erfassung des Leberfett- gehalts mittels digitaler quantitativer Analyse von B-Mode-Bildern erbrachten bisher nur mäßige Erfolge (ACORDA et al. 1994; BOBE et al. 2008). So konnte mit der Quantifizierung von Grauwertänderung als Diagnostikkriterium für die Fettleber eine Sensitivität von 38,5% und eine Spezifität von 88,5% (ACORDA et al. 1994), mit der Tiefendämpfung der Ultraschallwellen eine Sensitivität von 65,4% und eine Spezifität von 84,6%, und mit beiden eine Sensitivität von 38,5% und eine Spezifität von 100%
erreicht werden (ACORDA et al. 1994). Alternativ kann der Grad der Leberverfettung über eine zweistufige Berechnung aus digitalen Ultraschallparametern erfolgen (BOBE et al. 2008). Dabei wird zunächst der Grad der Leberverfettung unter Ver- wendung einer Diskriminanzfunktion geschätzt, und anschließend der TAG Gehalt innerhalb der ermittelten TAG Gehaltsgruppe mittels Regressionsanalyse bestimmt.
BOBE et al. (2008) verwendeten eine multiple lineare Kombinationen von 17 Echo- texturparametern der ersten und zweiten Grauwertstatistik, ohne diese näher zu be- schreiben. Bei der Unterteilung in vier Fettgehaltsstufen konnte der Leberfettgehalt unter Verwendung dieser 17 Echotexturparameter zu 82% vorhergesagt werden, bei zwei Stufen zu 92%. Die hohe Zahl verwendeter Echotexturparameter und die Tat- sache, dass für jede Leberfettgehaltsklasse eine eigene Formel benötigt wurde, ma- chen dieses Vorgehen allerdings sehr aufwendig und damit nicht praktikabel.
1.8. Fragestellungen und Zielsetzungen dieser Arbeit
Primäres Ziel dieser Arbeit war die Entwicklung und Etablierung einer nicht- invasiven, sonographischen Methode zur Bestimmung des Leberfettgehaltes bei Milchkühen.
Als Goldstandard für den Leberfettgehalt diente die biochemische Bestimmung von TL und TAG im Lebergewebe. Voraussetzung war somit, dass diese mit hoher Präzi- sion meßbar sind. In der verfügbaren Literatur lagen keine detaillierten Angaben be- züglich der Analysenmethoden zur Bestimmung von TL und TAG und deren Präzisi- on im bovinen Lebergewebe vor. Daher wurde zunächst eine solche Methode mit dem Ziel etabliert, dass diese auch in kleinen, aus Feinnadelschneidbiopsien (SCHOLZ et al. 1989) gewonnen Gewebemengen anwendbar ist (Kapitel 1).
Da bisher bei der digitalen Analyse von B-Mode Ultraschallbildern im Rahmen der Fettleberdiagnostik untersuchungs- und gerätebedingte Artefakte unberücksichtigt gelassen wurden, sollte geprüft werden, inwieweit deren Beachtung sensitivere und objektivere Ergebnisse liefert. Dafür wurde zunächst ein Computerprogramm entwi- ckelt, welches diese Korrekturen an B-Mode Ultraschallbildern vor der Bildanalyse durchführt (Kapitel 2). Die Software wurde dann an einer größeren Tierzahl auf ihre Eignung geprüft (Kapitel 3).
2. KAPITEL 1
Submitted
Tabellen und Abbildungen befinden sich am Ende des Manuskripts.
ANALYSIS OF TOTAL LIPID AND TRIACYLGLYCEROL CONTENT IN VERY SMALL LIVER BIOPSY SAMPLES IN CATTLE
Arbeitsanteil der Autoren an dem vorliegenden Manuskript
Initiative
Starke, Haudum, Busche, Rehage
Studiendesign
Haudum/Starke #, Busche, Rehage
Studiendurchführung
Praktische Untersuchungen Haudum, Starke
Analytik
Haudum, Busche, Wohlsein, Starke Statistische Auswertung
Haudum, Starke, Beyerbach, Dänicke, Rehage
Manuskripterstellung
Haudum/Starke #, Busche, Rehage
# gleichwertiger Beitrag von Haudum und Starke
Hepatic total lipid and triacylglycerol analysis
Analysis of total lipid and triacylglycerol content in very small liver biopsy samples in cattle1
A. Starke*,2,3, A. Haudum*,2, R. Busche*, M. Beyerbach†, S. Dänicke‡, J. Rehage*
* - Clinic for Cattle and
† - Institute for Biometry, Epidemiology and Information Processing,
University of Veterinary Medicine Hannover, 30173 Hannover, Germany
‡ - Institute of Animal Nutrition, Friedrich-Loeffler-Institute (FLI), Federal Research Institute for Animal Health, 38116 Braunschweig, Germany
1 This work was generously supported by a grant from the H. WILHELM SCHAUMANN STIFTUNG, Hamburg, Germany.
2 Both authors contributed equally
3 Corresponding author: alexander.starke@tiho-hannover.de
ABSTRACT
A procedure for analyzing total lipids (TL) and triacylglycerol (TAG) in two sequential steps using a small amount (100 mg) of bovine liver tissue is described. The TL was measured gravimetrically and TAG enzymatically in the TL extract, using an auto- mated analyzer. The TL analysis (n = 10; range, 40 – 314 mg/g wet fresh weight (FW)) yielded a mean CV of 2.2%. The TAG analysis (n = 10; range, 4 – 260 mg/g FW) yielded a mean intraday CV of 2.5% and a mean interday CV of 3.4%. The ana- lytical procedure was simple and accurate.
In a follow-up study, TL and TAG were measured in 150 German Holstein cows. The proportion of TAG relative to TL increased with increasing TL, to a content of ap- proximately 100 mg TL/g FW, and reached a plateau at approximately 68%. A linear, broken-line model, allowed accurate calculation of TAG from TL (r² = 0.98).
Key words: broken-line model, cattle, fatty liver, proportion of triacylglycerol relative to total lipid, small liver biopsy samples, triacylglycerol and total lipid determination
INTRODUCTION
Fatty liver is the most common liver disease in high-producing dairy cows in early lactation (Morrow, 1976; Roberts et al., 1981; Bobe et al., 2004). As the distribution of fat within the liver is homogeneous, small pieces of tissue are representative of the degree of fat infiltration (Gaal and Husveth, 1983). In research studies, the hepatic triacylglycerol (TAG) and total lipid (TL) contents are often determined quantitatively to characterize the severity of fatty liver (Selberg et al., 2005; Carlson et al., 2007;
Kalaitzakis et al., 2007). Based on a review of the literature, the amount of TAG rela- tive to TL varied greatly in fatty livers (Figure 1). Frome studies, liver samples with more than 100 mg TL/g wet fresh weight (FW) contained approximately 20 to 30%
TAG, whereas in other studies, the relative amount of TAG was 60 to 70%. Such dis- crepancies may reflect biological variance or differences in analytic precision. In most studies, detailed information regarding the precision of the methods was not given, and therefore, the reasons for the variation in the published proportions of TAG (Fig- ure 1) remained unclear.
- Figure 1 near here -
Until recently, at least 1 g of tissue was required to determine TL and TAG (Drackley et al., 1991; Veenhuizen et al., 1991; Bobe et al., 2008). Collecting such large sam- ples via transcutaneous liver biopsy was stressful, posed a health risk to the patient, and therefore appeared inappropriate for routine diagnostic purposes. Welfare con- siderations also call for minimally invasive techniques (Scholz et al., 1989).
Thus, the objective of this study was to develop a simple and precise analytical method for determination of hepatic TAG and TL in diseased and healthy cows, using small bovine liver samples (<100 mg FW). In a second part, the proportion of TAG relative to TL was determined in a large group of dairy cows. Another goal was to investigate whether the TAG content could be calculated reliably from the TL content, rather than being analyzed separately.
MATERIALS AND METHODS
The study was conducted under the guidelines of the Research Animal Act (research permit number 33.42502/06A372) of the Lower Saxony Federal State Office for Con- sumer Protection and Food Safety.
Part 1
Animals and procurement of liver biopsy samples for establishing analytical methodology
Ten German Holstein cows, which were (mean ± SD) 5.4 ± 2.2 yr old and weighed 576 ± 55 kg, were used. Approximately 10 g of hepatic tissue was collected from the caudal hepatic lobe intraoperatively during routine omentopexy for correction of left displacement of the abomasum (Dirksen, 1967). Analgesia for surgery was achieved by local paralumbar nerve blocks and infiltration of the incision line with approxi- mately 160 ml Isocaine 2%® (procainhydrochloride; Selectavet, Weyarn-Holzolling, Germany). Finadyne RP® (2.2 mg flunixin meglumine/kg BW; Essex Tierarznei, Mu- nich, Germany) was given as an analgesic before surgery and on the following day.
Small aliquots of liver biopsy samples were stored at -85 °C until further use. Re- peated measurements associated with establishing the analytical techniques neces- sitated collection of relatively large amounts of tissue.
Analysis
General method of analysis. For each analysis, separate liver aliquots thawed in a 40 °C water bath were used. Approximately 100 mg tissue was finely chopped using a scalpel blade. The TL content was determined gravimetrically, using a modi- fied method of lipid extraction (Hara and Radin, 1978). The lipid extract was resus- pended, divided into aliquots, and analyzed enzymatically for TAG content using a modification of a previously published technique (McGowan et al., 1983).
Determination of TL. As the amount of analyzed liver tissue was small, the
tec, SBC 21, Scaltec GmbH, Heiligenstadt, Germany) in a Teflon sealed screw-cap pyrex tube (Bibby Sterlin Ltd. Stone, England, UK). An aliquot (2 ml) of a 3:2 vol/vol mixture of hexane (95%; Baker, Deventer, the Netherlands) and isopropanol (Om- nichem, Bremen, Germany) were added and the TL was extracted in a rotator (La- bor- und Analysentechnik, Garbsen-Berenbostel, Germany) at 20 °C for 24 h. So- dium sulphate solution (1 ml, 0.455 mmol/l; AppliChem, Darmstadt, Germany) was added and the samples were vortexed (Ing. Büro Cat, M. Zipperer GmbH, Staufen, Germany) and then centrifuged (Heraeus Sepatech, Themo Electron, Langenselbold, Germany) at 400 g for 10 min at room temperature. The supernatant, which con- sisted of the hexane phase with dissolved lipids, was removed and placed in empty pre-weighed reagent tubes (Sarstedt, Nürmbrecht, Germany). For complete lipid ex- traction, 1 ml of hexane was added again, the sample was vortexed and centrifuged, and the supernatant removed and added to the previously collected supernatant.
Hexane was evaporated by placing the supernatant in a vacuum centrifuge (Eppen- dorf AG, Hamburg, Germany) for 2 h. The TL content of the analyzed liver sample was determined gravimetrically in mg per gram of liver FW; this was done by sub- tracting the weight of the empty tube from the weight of the tube containing the ex- tracted lipid, considering the original amount of fresh hepatic tissue.
Determination of TAG. After TL analysis, 1 ml of hexane was added to the lipid extract (range, 9 to 75 mg) to determine TAG content. To prevent premature satura- tion of the enzymatic measurement system (measurement range of the test kit: 0 to 13 mmol/l (0 – 11.4 mg/ml) TAG), the volume of the hexane-lipid mixture used for analysis was adjusted to the amount of the lipid extract. Thus, for <15 , 15 – 35, and
>35 mg lipid extract, 40, 20, and 10 μl of hexane-lipid mixture was analyzed, respec- tively (Hamilton Mikropipettierspritze, Mikroliter™ Mikroliter®#75, Hamilton, Bonaduz, Switzerland) using reagent tubes. The hexane was removed by evaporation in a fume hood.
The lipid extract was taken up in 80 μl buffer, 48.1 mmol/l disodium hydrogen phos- phate (Na2HPO4, pH 7, adjusted using 5 M hydrochloric acid) with 1 μmol/l nonaethylene glycol monododecyl ether detergent (Lubrol, Sigma, Steinheim, Ger-
many). Triglyceride reagent (100 μl; microbial lipase, Sigma, Steinheim, Germany) and double-distilled water (420 μl) were added and the samples were incubated overnight for 16 h at 37 °C (Thermomixer Eppendorf-Thermomixer comfort, Eppen- dorf AG, Hamburg, Germany) in order to ensure complete lipolysis of TAG. The TAG content of the sample was then measured enzymatically (McGowan et al., 1983) with an automated analyzer (ABX Pentra 400, HORIBA ABX, Montpellier, France), using the Triglyceride Mono-Fluid GPO PAP Kit (mti diagnostics GmbH, Idstein, Germany) according to the manufacturer’s instructions, except that the measured sample size was 12 μl instead of the suggested 3 μl. The TAG concentration in liver tissue was calculated from the result displayed by the automated analyzer (mmol/l) and ex- pressed in mg per gram of liver FW, based on an average molecular mass of 875 U (Anonymus, 1981).
Quality controls. As a standard for TAG, triolein (5 g TAG/l; Sigma, Steinheim, Germany) in concentrations of 0.167, 0.5, and 1.5 g TAG/l, reflecting the lower, aver- age and upper ranges, respectively, of the TAG content in liver samples, was used.
Standards were analyzed in parallel with the hepatic TAG analysis. Triolein stan- dards were processed in the same way as the TAG analysis of lipid extracts from liver samples described above. Accuracy of TAG determination was tested by calcu- lating the intraday and interday CV and the recovery of triolein standards. The intra- day CV was determined by measuring tissue samples (n = 10) five times consecu- tively on the same day. The interday CV was determined by measuring tissue sam- ples (n = 10) on four different days within 3 wk.
Modifications of TAG analysis. Modifications of the TAG analysis method de- scribed above were tested using various detergents, lipases, incubation periods, and lipase storage conditions. To determine the optimal pre-incubation period of lipid ex- tracts for complete lipolysis, four samples of aliquoted lipid extracts of one liver sam- ple were incubated with microbial lipase at 37 °C (Thermomixer) for 0.5, 1, 2, 4, and
quots of triglyceride reagent were stored at -20 °C and 4 °C for 3 wk. After this time, the lipolytic activity of each lipase (four samples) was evaluated in one liver sample.
Instead of microbial lipase (triglyceride reagent) also A) Porcine pancreatic lipase (7.7 × 104 U/l; Lipase from porcine pancreas type II, Sigma, Steinheim, Germany) and B) Porcine pancreatic lipase (7.7 × 104 U/l; Lipase type VI-S: from porcine pan- creas, Sigma, Steinheim, Germany), were tested. In combination with the buffer solu- tion (48.1 mmol/l Na2HPO4), 10% SDS solution (337 mmol/l; AppliChem Darmstadt, Germany) was tested as an alternative for the detergent Lubrol. The pH of the buffer solution was adjusted to 8.0 for the pancreatic and to 7.0 for the microbial lipases, using 5 M hydrochloric acid. Each detergent was assessed three times for each of the pancreatic and the microbial lipases.
Part 2
Animals and procurement of liver biopsy samples
Liver biopsies (approximately 1,000 mg) from the caudal lobe of 150 German Hol- stein cows (mean ± SD; age: 4.9 ± 2.0 yr, BW: 571 ± 80 kg) from 140 farms were taken during laparotomy for correction of a left-sided abomasal displacement, as de- cribed above. Approximately half of each sample (500 mg) was either stored at - 85 °C until analysis of TAG and TL, or was fixed in formaldehyde.
Histopathologic examination
Formaldehyde-fixed tissues were embedded in paraffin, sectioned and stained with haematoxylin and eosin and Sudan III stain for histopathotologic evaluation. Exami- nation of sections to exclude hepatic diseases other than hepatic lipidosis (Mertens, 1992) was performed by light microscopy (objective-magnification 4× - 40×; BX60, Olympus, Tokyo, Japan).
Analysis of TL and TAG
Analysis of hepatic TL and TAG was performed as described for Part 1 of the present study. Analyses of TL and TAG were carried out three and five times, respectively.
For data analyses, the means of TL and TAG measurements were used.
Statistical analyses
The program SAS (release 9.1 for Windows, SAS Institute Inc., Cary, N.C., USA) was used for statistical analysis. Means ± SD and CV were calculated for all results (pro- cedure PROC MEANS). In Part 2, TAG, the difference between TL and TAG, and the proportion of TAG relative to TL (proportion = TAG/TL * 100) were regressively re- lated to TL, using either the simple linear regression model or the linear broken-line model. Data were fitted to the models according to SAS procedures (procedure PROC NLINE) published by Robbins et al. (2006). The latter approach assume a so- called break-point (BP) and implied that the slope of the regression line for xy-pairs with x-values was lower than the BP was different from the slope for values greater than the BP. Both models were compared with regard to the adjusted r² and residual standard deviation (RSD). Moreover, the measured TAG contents were plotted against the residuals of predicted TAG contents in percentage of measured TAG, to enable an evaluation of the predictability of both models. SigmaPlot® 2001 (Systat Software Inc., Chicago, IL, USA) was used to create graphs.
RESULTS Part 1
TL
The results of TL determination in the ten tissue samples are shown (Table 1). These samples were analyzed three times, and the maximum and mean CV were 4.5 and 2.2%, respectively.
- Table 1 near here -
TAG
Measuring TAG using the established method. The mean recovery rate of TAG in the triolein standards (low, average, high) conducted on four days was 100.7 ± 0.3%, the intraday CV < 1.1%, and the interday CV < 3.0% (Table 2). The intraday and interday CV of the measurement of TAG in ten liver tissue samples on four days (means ± SD from five individual measurements) were <3.4% (Table 2).
- Table 2 near here -
Evaluation of detergent and lipase. With all three lipases, the detergent SDS (337 mmol/l; incubation 4 h) yielded a maximum recovery rate of 15% of the amount of TAG in the triolein standard. However, the detergent Lubrol (1 μmol/l; incubation 4 h) yielded a mean recovery rate of 90% for the microbial lipase, and a maximum recovery rate of 25% for both of the pancreatic lipases.
Duration of lipase incubation and storage temperature of lipase. The recov- ery rate of TAG in the triolein standard increased with increasing duration of incuba- tion, up to 10 h (Figure 2). After incubation for 4 h, the mean recovery rate was 90.5 ± 3.2%, and after 10 h, it was 100.3 ± 2.6%. There was no difference between the activity of microbial lipase stored at -20 °C and 4 qC for 3 wk (Figure 2). For prac- tical reasons, the incubation period was 16 h (overnight).
- Figure 2 near here -
Part 2
For 150 liver tissue samples, TL contents ranged from 32.8 to 395.7 mg/g FW, with a mean CV of <2.8%. The TAG content ranged from 4.5 to 292.4 mg/g FW and the mean CV was <1.5%. Histologic examination of the 150 liver biopsy samples re- vealed no abnormal histologic findings other than hepatic lipidosis.
There was an apparent linear increase in TAG content with an increase in the TL content (Figure 3). Based on the slope of the regression line, there was an increase of 0.75 mg TAG for an increase in TL by 1 mg. The r² was 0.97. Thus, whereas ap- proximately 97% of the variance of the data overall were explained by this simple linear relationship, it was apparent (Figure 4) that the accuracy of prediction of the TAG content, based on TL, decreased with TL contents lower than 80 to 100 mg/g FW (Figure 3), corresponding to TAG contents lower than 50 mg/g FW. A closer ex- amination of the relationship between TL and TAG suggested slightly different linear slopes for xy-pairs below and above the BP. The existence of a BP becomes appar- ent when the difference between TL and TAG (Figure 7) and the proportion of TAG relative to TL (Figure 8) were plotted against TL. Therefore the data were fitted to the broken-line model (Figures 5, 7, 8), which slightly increased the variance accounted by the model (r² = 0.98; Figure 5) and increased the accuracy of prediction of TAG based on TL, as indicated by a lower variation of the residues in the lower range of the TL content (Figure 6). The relationship between TL and TAG was characterized by a slope of 0.95 in the lower range and a slope of 0.7 in the higher range. Both slopes were discriminated from each other by the fitted BP at 107 mg TL/g FW. The slopes and BP for the relationships between the difference of TL and TAG, and the proportion of TAG relative to TL, are shown (Table 3). Above the BP (>82 mg TL/g FW), the proportion of TAG relative to TL increased only slightly, with increasing TL contents (slope, 0.01); the proportion reached a plateau at approximately 68% (Fig- ure 8). The curve representing the relationship between the difference between TL
to the BP (107 mg TL/g FW), and a more pronounced increase beyond the BP (slope, 0.3; Figure 7).
- Figure 3 – 8, Table 3 near here -
DISCUSSION
Depending on the lactation stage, lactation number, feeding regime and energy level of the ration TL and TAG contents of approximately 40 – 300 and 10 – 200 mg/g FW, respectively, have been measured in bovine liver samples (Brumby et al., 1975; Reid et al., 1977; Reid, 1980; Gaal et al., 1983; Gerloff and Herdt, 1984; Herdt, 1988; Jor- ritsma et al., 2001). The liver biopsy samples used in the present study were pro- cured from cows with normal to severe fatty infiltration of the liver (TL: 32 – 396 mg/g FW; TAG: 4 – 293 mg/g FW). Therefore, the selection of samples represented the expected range and appeared ideal for establishing and testing the analytical meth- ods.
One of the goals of the present study was to investigate the feasibility of using no more than 100 mg of liver tissue to comply with animal welfare regulations, as well as to reduce the time and labour involved with procurement and evaluation of samples.
Methods used in previous studies required up to 1,000 mg of liver tissue for deter- mining the TL content (Hara and Radin, 1978; Gaal et al., 1983; Kalaitzakis et al., 2007) and up to 10,000 mg of tissue for TAG (Bulter et al., 1961; Bickhardt et al., 1988). Resuspension and further processing of the lipid extract after TL determina- tion allowed a substantial reduction of the amount of tissue required for analysis.
Studies determining the TL and TAG content in two sequential steps using one liver biopsy sample required 1,000 to 3,000 mg of tissue (Drackley et al., 1991; Veen- huizen et al., 1991; Bobe et al., 2008). Gaal et al. (1983) were the only authors to report a method that required only 100 mg of bovine liver tissue, although there was no detailed description of the methods used, nor critical assessment of the precision and accuracy of the results. Therefore, we were unable to implement that method in our study. Rodriquez-Sudera and Peinado-Onsurbe (2005) described in detail a method for extracting TL and determining of TAG in two sequential steps, using 50 mg of rat and mouse liver tissue. However, the TAG concentration varied greatly and four measurements in duplicate yielded CV ranging from 11 to 43%. Analytical problems and the various extraction and buffer solutions and detergents were cited
tempted to improve the reliability and accuracy and the analytical methods, and thus modified various published methods.
For instance, for TL determination the extraction method and subsequent gravimetric measurement technique described by Hara and Radin (1978) were modified. These authors used hexane/isopropanol rather than chloroform-methanol (Folch et al., 1957; Atkinson et al., 1972) for separating of the water soluble and lipid components of tissue. Hexane-isopropanol provides more rapid and precise separation of the lipid and non-lipid phases and is less of a health hazard compared with chloroform- methanol (Anonymus, 1973). However, Hara and Radin (1978) used 1,000 mg of rat or mouse brain tissue in their study. In our study, approximately 100 mg of bovine liver tissue was used in an effort to develop an accurate technique using only a small amount of tissue. To ensure complete lipid extraction from a small tissue sample, a second extraction step using hexane was done. Based on Parts 1 and 2, a reduction in the amount of tissue had no negative effect on the accuracy of the results, neither in the low nor in the high ranges of TL (Table 1). Most studies did not report the accu- racy of their results, and thus comparison with our results was not possible (Folch et al., 1957; Hara and Radin, 1978). In one study (Atkinson et al., 1972), a mean CV of approximately 1% was reported for lipid extraction from subcutaneous fat tissue.
To determine the TAG content, the TL extract was resuspended in hexane, and ali- quots of various volumes were created, depending on the amount of the available extract. In contrast to the results of a previous study (Rodriquez-Sureda and Peinado-Onsurbe, 2005) the lipid extract was completely dissolved in hexane. The TAG content was subsequently evaluated colorimetrically, using an automated ana- lyzer (McGowan et al., 1983). The latter method previously produced accurate and reproducible results (Winartasaputra et al., 1980; McGowan et al., 1983) compared with fluorometric analysis (Winartasaputra et al., 1980). As the lipases in the serum test kits used in the present study did not provide complete hydrolysis of TAG, prein- cubation with another lipase was carried out, which provided complete lipolysis of TAG (Table 2). The recovery rate of the methods used was determined by running controls with triolein as the TAG standard, which has been used in several human medical and veterinary studies (Fletcher, 1968; Hamilton et al., 1983; Rodriquez-
Sureda and Peinado-Onsurbe, 2005). Based on the results of the triolein standard (Table 2), incubation with microbial lipase afforded complete lipolysis of TAG (recov- ery rate of the triolein standard: 100.3 ± 2.6%). The 10 min incubation period re- ported by Sugiura et al. (1977), Megraw et al. (1979), Winartasaputra et al. (1980) and McGowan et al. (1983) was inadequate for hydrolysis of TAG. Incubating the samples for 16 h overnight was the most practical interval.
The methods used had a high level of precision; the intraday CV was <3.4% and the interday CV was <3.4%. These CV also considered potential possible variations caused by creation of aliquots from the lipid extract. In addition to using a smaller amount of tissue, our method was also associated with an increase in accuracy com- pared with results reported by Rodriquez-Sudera and Peinado-Onsurbe (2005; CV 1.5% vs. 11.3%). This was confirmed in the second part, in which liver biopsy sam- ples from 150 cows were analyzed.
Based on analysis of the 150 liver biopsy samples, the TAG content increased with an increase in TL content, which was in agreement with other studies (Brumby et al., 1975; Reid et al., 1977; Staufenbiel et al., 1992, 1993; Carlson et al., 2006; Kalait- zakis et al., 2006; Carlson et al., 2007; Kalaitzakis et al., 2007; McFadden et al., 2008). The relationship between TAG and TL content was characterized with suffi- cient accuracy by a linear function (r² = 0.97; RSD = 11.2), and the TAG content could be calculated from the TL, within the range of 32 – 396 mg TL/g FW. The es- tablishment of this relationship eliminated the need to determine the TAG content for routine diagnostic workups. However, in the lower range of TL content, there was a noticeable difference between the measured and calculated TAG content (Figure 4).
To overcome this drawback, the linear broken-line regression model was used. This model assumed the existence of a BP, which assigned different slopes for corre- sponding data pairs with x-values below and above the BP. The resulting new re- gression line diminished the difference between measured and calculated TAG in the lower range of TL.
Graphs depicting the relationship between TL and TAG revealed a deviation in the
ous studies, except in one on rats with hepatic lipidosis induced by a high-fat diet, in which there was a non-linear increase in the TAG content of the liver (Gauthier et al., 2004). In Part 2 of the present study, the change in linear relationships between TAG and TL at a certain level of TL, was most obvious when the difference between TL and TAG and the proportion of TAG relative to TL were plotted against TL. In agree- ment with published reports (Gaal et al., 1983; Herdt et al., 1983; Reid and Roberts, 1983; Staufenbiel et al., 1993), there was an initial increase in the proportion of TAG relative to TL. However, this was apparent only up to a TL content of approximately 70 to 120 mg/g FW; above this level, the proportion of TAG reached a plateau. Using the broken-line model, the BP, which discriminated between lower and higher TL contents, could be calculated. The occurrence of a BP in the linear relationship be- tween the two variables was intriguing. Although the scatter plot (Figure 1) suggested a possible change in the relationship between the proportion of TAG and TL at a cer- tain TL level, this has not previously been reported for cattle. Except for one study (61%; Staufenbiel et al., 1992) the reported proportions of TAG relative to TL were considerably lower (13.1 – 49.1%) in the higher TL range than the ones reported here. A possible reason for this could be more complete hydrolysis, and thus a higher TAG recovery rate in our study. Incomplete enzymatic breakdown of TAG at levels of 100 mg TL/g FW and higher, because of too high a substrate concentration, can be excluded as an explanation for the observed plateau; the measuring range of the test kits was 0 to 11.4 g TAG/l. Furthermore, the use of aliquots of the hexane-lipid mix- ture limited the amount of TAG to 1.4 g/l.
The phenomenon of the linear broken-line relationship between the proportion of TAG relative to TL vs. the TL remains to be clarified. It was noteworthy that the pro- portional amount of TAG relative to TL reached a plateau at approximately 70%, with a BP of 70 to 120 mg/g FW TL, and henceforth refrained from increasing any further (Figure 8). The proportional amount of remaining lipids (TL minus TAG) relative to TL decreased continuously with increasing TAG, until the BP was reached, but thereaf- ter no longer. Nevertheless, the TL and TAG values still increased further after this point (Figure 5). The TAG content rose less steeply with increasing TL after BP was reached (slope 0.70 mg TAG per mg TL, in contrast to former slope 0.95 mg TAG per
mg TL; Figure 5). The absolute amount of remaining lipids stagnated until BP was reached (Figure 7), only to increase thereafter with rising TL content (slope 0.30 mg remaining lipids per mg TL; Figure 7). After the BP was reached (82 mg TL/g FW;
Figure 8; or 107 mg TL/g FW; Figures 5 and 7), TAG was deposited at a constant proportion relative to TL (Figure 8). It appeared that a saturation equilibrium limited re-esterification of NEFA to TAG at a certain TL value (approximately 100 mg/g FW).
Irrespective of the further increase in TAG content, this equilibrium presumably lead to an increase in the absolute amount of other lipid fractions when the TL exceeded approximately 107 mg/g FW (Figure 5). These substances likely included NEFA, cho- lesterol and cholesterol esters, because the content of phospholipids changed little during excessive fatty infiltration of the liver (Brumby et al., 1975; Reid et al., 1977).
The exact reasons and causes for the occurrence of the phenomenon of the linear broken-line relationship between the TAG and the proportion of TAG relative to TL, or rather the occurrence of a saturation equilibrium at a certain TL content, remain speculative and require further studies.
CONCLUSION
The method described in this report enabled the determination of TL and TAG in two sequential steps using one sample of liver tissue (50 to 100 mg) procured via a commercial cutting biopsy needle. The method is straightforward, precise and accu- rate. The relationship between TAG and TL content at various severities of fatty infil- tration could be characterized for the first time using a linear broken-line model with a BP at 107 mg TL/g FW. This allowed the reliable calculation of TAG (r² = 0.98) from liver tissue with a wide range of TL content (32 to 396 mg TL/g FW).
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Table 1: Gravimetric measurement of total lipid (TL) content in liver biopsy speci- mens from ten German Holstein cows
Cow Total lipid content (mg/g FW1) CV (%)
Analysis
1st 2nd 3rd Mean ± SD
1 42.2 42.6 41.2 42.0 ± 0.7 1.7
2 55.6 55.1 52.9 54.5 ± 1.4 2.6
3 60.8 60.3 59.1 60.1 ± 0.9 1.5
4 122.7 123.2 122.6 122.8 ± 0.3 0.3
5 167.4 173.0 170.3 170.2 ± 2.8 1.7
6 182.5 170.5 185.8 179.6 ± 8.0 4.5
7 179.8 184.7 187.2 183.9 ± 3.8 2.0
8 183.6 195.6 186.2 188.5 ± 6.3 3.3
9 195.0 197.4 192.6 195.0 ± 2.4 1.2
10 314.4 323.3 305.5 314.4 ± 8.9 2.8
Range 42.0 – 314.4 0.3 – 4.5
MeanCV 2.2
1 FW = wet fresh weight
ean + SD; five analyses) of triacylglycerol (TAG) analysis on four consecutive days, recovery rate (%), and CV; olein; 5 mg TAG/ml; Sigma, Steinheim, Germany) and in hepatic tissue from analysis Mean over all days 2 3 4 analysis Intraday CV (%) Mean ± SD (mg/ml) Intraday CV (%) Mean ± SD (mg/ml) Intraday CV (%) Mean ± SD (mg/ml) Intraday CV (%) Mean ± SD (mg/ml) Interday CV (%) 0.8 0.17 ± 0.0010.6 0.16 ± 0.0010.5 0.17 ± 0.002 1.0 0.17 ± 0.005 2.9 1.1 0.50 ± 0.0010.3 0.52 ± 0.0030.6 0.48 ± 0.003 0.7 0.50 ± 0.015 3.0 1.0 1.50 ± 0.0090.6 1.53 ± 0.0140.9 1.50 ± 0.012 0.7 1.51 ± 0.016 1.1 rate Mean ± SD (%) Mean ± SD (%) Mean ± SD (%) Mean ± SD (%) 102.2 ± 0.7 97.3 ± 0.5 104.1 ± 1.1 100.8 ± 0.8 99.8 ± 0.3 103.4 ± 0.6 96.4 ± 0.7 100.3 ± 3.0 99.9 ± 0.6 102.1 ± 1.0 100.0 ± 0.8 100.9 ± 0.8 nalysis 4 ) Intraday CV (%) Mean ± SD (mg/g FW) Intraday CV (%) Mean ± SD (mg/g FW) Intraday CV (%) Mean ± SD (mg/g FW) Intraday CV (%) Mean ± SD (mg/g FW) Interday CV (%) 5.2 14.5 ± 0.4 2.4 15.2 ± 0.3 1.9 15.3 ± 1.1 7.3 14.7 ± 0.6 3.9 1.8 18.4 ± 0.2 1.3 19.4 ± 0.6 3.0 18.6 ± 0.6 3.0 18.9 ± 0.4 2.3 0.4 21.9 ± 0.7 3.0 23.2 ± 0.8 3.4 21.2 ± 1.2 5.6 21.9 ± 0.9 4.0 2.8 90.1 ± 0.7 0.8 93.8 ± 1.3 1.3 94.9 ± 1.2 1.3 92.9 ± 2.5 2.7 1.3 118.3 ± 4.5 3.8 117.2 ± 2.2 1.9 120.5 ± 3.0 2.4 117.3 ± 3.2 2.7 1.4 138.4 ± 3.7 2.7 145.6 ± 2.5 1.7 134.5 ± 6.8 5.1 139.8 ± 4.6 3.3 0.8 129.8 ± 1.0 0.8 131.6 ± 2.6 2.0 139.3 ± 3.1 2.3 134.1 ± 4.3 3.2 0.5 118.3 ± 1.5 1.3 105.9 ± 3.1 2.9 117.2 ± 3.4 2.9 114.0 ± 5.6 5.0 3.3 163.1 ± 2.8 1.7 159.4 ± 3.4 2.1 162.5 ± 2.8 1.8 159.4 ± 4.8 3.0 3.0 263.9 ± 3.6 1.4 246.3 ± 2.8 1.2 249.6 ± 4.6 1.8 250.4 ± 9.8 3.8 2.0 1.9 2.1 3.3 3.4
low = 0.167 g/l TAG, triolein standard diluted 1 : 30 average = 0.5 g/l TAG, triolein standard diluted 1 : 10 high = 1.5 g/l TAG, triolein standard diluted 1 : 3.33 FW = wet fresh weight
