Am.  Jour.  Pharm.  ) 
July,  1915.  X 
The  Estimation  of  Fat. 
309 
THE  ESTIMATION  OF  FAT.1 
By  Helman  Rosenthal  and  P.  F.  Trowbridge, 
From  the  Department  of  Agricultural  Chemistry  of  the  University  of 
Missouri,'  Columbia. 
In  the  course  of  the  nutrition  investigations  in  this  laboratory  we 
have  occasion  to  make  a  great  many  estimations  of  fat  in  samples  of 
portions  of  animals  and  in  feeding  stuffs.  The  usual  Soxhlet  extrac- 
tion of  the  thoroughly-dried  sample  with  ether  is  the  method  that 
has  been  employed.  It  is  understood  that  this  method  gives  a  deter- 
mination of  the  crude  fat  or  total  ether-soluble  material.  By  the 
Soxhlet  extraction  of  dried  blood  we  have  been  unable  to  find  any 
ether-soluble  substances.  It  has  been  very  difficult  to  obtain  con- 
cordant results  with  such  material  as  brain,  liver,  and  bran,  and  it 
has  been  found  that  sixteen  hours'  extraction  of  these  substances 
would  not  remove  all  the  ether-soluble  material.  A  reextraction 
frequently  adds  a  very  appreciable  amount  of  crude  fat.  It  is 
because  of  these  difficulties  that  we  have  investigated  the  methods 
proposed  for  accurate  fat  estimation  in  the  hope  that  we  could  find 
a  method  that  would  give  a  true  determination  of  the  fat  present,  and 
at  the  same  time  permit  application  in  a  laboratory  where  a  great 
many  determinations  must  be  made. 
Kumagawa  and  Suto  2  have  made  a  study  of  the  general  methods 
of  fat  estimation.  The  methods  proposed  by  Rosenfeld,  by  Dor- 
meyer,  and  by  Glickin  are  also  extraction  methods,  and  therefore 
are  no  more  accurate  than  the  regular  Soxhlet  method.  Kumagawa 
and  Suto  saponify  the  fat  with  5N  sodium  hydroxide  solution.  They 
transfer  the  soap  to  a  separatory  funnel  and  acidify  with  a  mineral 
acid.  The  fatty  acids  are  shaken  out  with  ether,  the  ethereal  solution 
is  evaporated  to  dryness,  taken  up  with  ether,  and  filtered  through 
asbestos.  The  filtrate,  after  being  dried,  is  taken  up  with  petroleum 
ether  and  again  filtered.  The  filtrate  is  evaporated  and  dried  to 
constant  weight.  The  residue  is  taken  up  with  petroleum  ether  and 
treated  with  alcoholic  potash.  The  petroleum  ether  is  removed  and 
the  alcoholic  solution  is  again  shaken  with  petroleum  ether.  These 
combined  ether  extracts  are  evaporated  and  taken  up  with  a  little 
alcoholic  sodium  hydroxide.     The  solution  is  evaporated,  dried, 
1  Reprinted  from  Journal  of  Biological  Chemistry. 
2  M.  Kumagawa  and  K.  Suto :  Biochem  Ztschr.,  iii,  pp.  212-437,  1908. 
