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The application of high pressure liquid chromatography to the analysis of clinically important porphyrins Carlson, Robert Eric 1976

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THE APPLICATION OF HIGH PRESSURE LIQUID CHROMATOGRAPHY TO THE ANALYSIS OF CLINICALLY IMPORTANT PORPHYRINS  by  ROBERT ERIC CARLSON B.A., University of Minnesota-Duluth, 1972  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of CHEMISTRY  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA July, 1976  (c)  Robert E r i c Carlson, 1976  In p r e s e n t i n g t h i s  thesis  in p a r t i a l  fulfilment of  the requirements f o r  o  an advanced degree at the I  Library shall  f u r t h e r agree  for  make i t  freely available  that permission  for  I agree  r e f e r e n c e and  f o r e x t e n s i v e copying o f t h i s  that  study. thesis  s c h o l a r l y purposes may be granted by the Head of my Department or  by h i s of  the U n i v e r s i t y of B r i t i s h Columbia,  this  written  representatives. thesis  is understood that copying or p u b l i c a t i o n  f o r f i n a n c i a l gain s h a l l  permission.  Department of The  It  TnJTS r  University of B r i t i s h  Columbia  2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5  Date  '  2Q  jL4>  .  /?  f-C  not be allowed without my  i  ABSTRACT The porphyrias are a group of acquired or inherited metabolic diseases characterised by the abnormal production and excretion of porphyrins and porphyrin precursors. sequent treatment  The c l i n i c a l evaluation and sub-  of porphyria are dependent on the analysis of these  compounds i n urine, feces and blood because a wide array of overlapping physiological manifestations make d i r e c t c l i n i c a l diagnosis d i f f i c u l t . The a n a l y t i c a l techniques currently a v a i l a b l e are i n e f f i c i e n t and do not have the capacity to separate or e a s i l y quantify complex mixtures of porphyrins.  High pressure l i q u i d chromatography (HPLC), on the other  hand, can readily achieve complex separations and since the sample being analyzed remains i n solution at a l l times i t s detection and quantitation are considerably s i m p l i f i e d . Bearing these considerations i n mind, we have developed  new  porphyrin i s o l a t i o n techniques and evaluated a variety of HPLC packing/ solvent systems for the analysis of the uro to proto porphyrins from urine and feces.  The HPLC procedures  are rapid and e f f i c i e n t and should be  e a s i l y extended to other porphyric samples.  The v e r s a t i l i t y of the HPLC  system was demonstrated by the characterization of copper coproporphyrin from a porphyric f e c a l sample.. We have also developed  procedures  for the q u a l i t a t i v e analysis of  the f e c a l sub-uro porphyrins and made prelminary progress toward the separation and characterization of this heterogeneous group of compounds.,  ii  CONTENTS  SECTION  PAGE  Abstract  i  Contents  ' . . • .•  1  L i s t of Tables  v  L i s t of Figures  v  Acknowledgements Dedication -  INTRODUCTION A.  B.  i  .viii •  %  I.  1  l  x  1  C l i n i c a l and biochemical aspects of porphyria  1  1.  Porphyrin metabolism a. Porphyrin structure b. Porphyrin biosynthesis c. Porphyrin excretion  1 2 5 7.  2.  C l a s s i f i c a t i o n of the porphyrias: biochemical symptoms  C l i n i c a l and  Current " a n a l y t i c a l methods for the determination of porphyrins and porphyrin precursors i n c l i n i c a l samples  9  14  1.  Urine samples a. Porphyrin precursors b. Uro to proto porphyrins  14 14 14  2.  Fecal samples a. Uro to proto porphyrins b. Sub-uro porphyrins  15 15 18  3.  Blood samples  19  CONTENTS, continued.  iii  SECTION  PAGE  C. . HPLC background  II.  21  1.  Resolution theory  21  2.  Separation modes  22  3.  Porphyrin HPLC  22  EXPERIMENTAL A.  24  HPLC instrumentation  24  1.  The high pressure l i q u i d chromatograph  24  2.  Special apparatus:  A variable wavelength and  scanning detector B.  C.  D.  25  Solvent and column preparation  26  1.  Solvents  26  2.  Columns  26  Porphyrin i d e n t i f i c a t i o n  and quantitation procedures  ... 28  1.  Porphyrin standards  28  2.  Stop-flow v i s i b l e spectra  28  3.  Mass spectra  28  4.  Quantitation  29  Pre-analysis preparation of porphyric samples  28  1.  Urine samples  29  2.  Fecal samples  30  E.  Spectroscopic analysis of the sub-uro f r a c t i o n  32  F.  HPLC of c l i n i c a l samples  33  1.  33  Urine samples  CONTENTS, continued.  iv  SECTION  PAGE 2.  G.  H. III.  I d e n t i f i c a t i o n of the " t r i c a r b o x y l i c acid porphyrins"  B.  C.  33 33 33 .. 34  1.  Analogues of harderoporphyrin  34  2.  Copper coproporphyrin  34  I d e n t i f i c a t i o n of the sub-uro porphyrins  RESULTS A.  IV.  Fecal samples a. Uro to proto porphyrins b. Sub-uro porphyrins  35 36  Investigation of high pressure l i q u i d chromatographic parameters f o r the optimization of porphyrin analysis  36  1.  Uro to proto porphyrins a. Porphyrin free acids b. Porphyrin esters i. Adsorption chromatography ii. Gel (permeation) chromatography  36 36 37 37 43  2.  Sub-uro porphyrins a. Adsorption chromatography b. Gel (permeation) chromatography  44 44 45  Sample preparation procedures  45  1.  Urine samples  45  2.  Fecal samples  46  Sample analysis  47  1.  Urine samples  47  2.  Fecal samples a. Gel chromatographic analysis b. Uro to proto porphyrins c. Sub-uro porphyrins i. Screening procedures ii. Chromatography and i d e n t i f i c a t i o n  50 50 52 57 57 58  CONCLUSION  BIBLIOGRAPHY  62 65  V  LIST OF TABLES TABLE  PAGE  I  Porphyrins referred to i n this study  4  II  Porphyrins found i n porphyric samples  8  III  C l a s s i f i c a t i o n of the porphyrias  9  IV  Main c l i n i c a l features of the porphyrias  V  C l i n i c a l features of the acute attack: percentage incidence  VI  VII VIII VIII  10 A comparison of 11  Values for urinary, f e c a l and erythrocyte porphyrins and porphyrin precursors i n porphyric and normal patients  13  Description of the HPLC column packings used i n this study  27  a. A comparison of the effect of ethyl acetate and ethyl propanoate on porphyrin retention (k')  40  b.  A comparison of the r e l a t i v e v a r i a t i o n i n retention (k') with propyl, ethyl and methyl porphyrin esterification  IX  Results of the HPLC analysis of porphyric urine  X  Results of the HPLC analysis of porphyric feces uro to proto porphyrins  40 47  54  XI  Copper coproporphyrin test samples  55  XII  Spectroscopic analysis of the sub-uro porphyrin f r a c t i o n by the column and Rimington procedures  57  vi  LIST OF FIGURES FIGURES  PAGE  1.  Uroporphyrinogen  2.  Regions of the heme b i o s y n t h e t i c pathway a f f e c t e d i n the p o r p h y r i a s  3a.  I I I metabolism  6  12  TLC a n a l y s i s o f a c i d / e t h e r e x t r a c t e d p r o t o and copro f r a c t i o n s from p o r p h y r i c f e c e s  16  3b.  TLC/chromatoscan chromatograph  4.  Schematic o f an HPLC system  24  5.  Diagram o f the HPLC/Cary 17 flow c e l l  25  6.  P r o t o p o r p h y r i n IX on a C-18 r e v e r s e phase p a c k i n g  37  7.  P r o t o p o r p h y r i n IX d i m e t h y l e s t e r (2) and c o p r o p o r p h y r i n I I I t e t r a m e t h y l e s t e r (4) on a b a s i c alumina packing  38  U r o p o r p h y r i n I I I (8) to c o p r o p o r p h y r i n I I I (4) methyl e s t e r s on a n e u t r a l alumina p a c k i n g  38  8.  9.  Uroporphyrin  I I I octamethyl  o f a p o r p h y r i c sample  17  e s t e r on a P o r a s i l C  packing  39  10.  I l l u s t r a t i o n o f the s o l v e n t g r a d i e n t  11a.  Reference chromatogram o f uro t o p r o t o p o r p h y r i n (8-2) methyl e s t e r s on C o r a s i l I I  42  lib.  Reference chromatogram of uro t o p r o t o p o r p h y r i n (8-2) methyl e s t e r s on P o r a s i l T  43  12.  S e p a r a t i o n o f uro ( 8 ) , copro  (254 nm)  41  (4) and p r o t o p o r p h y r i n (2)  m e t h y l e s t e r s on Sephadex LH-20  44  13a.  Chromatogram o f u r i n e sample 5 (Table IX)  47  13b.  Chromatogram o f u r i n e sample 4 (Table IX)  48  14.  Comparison of the HPLC and l i t e r a t u r e a n a l y s i s o f Symptomatic and C o n g e n i t a l e r y t h r o p o i e t i c p o r p h y r i a samples  49  HPLC g e l chromatograms of s e l e c t e d f e c a l methylene' dichloride extracts  51  15.  vii FIGURES, continued. FIGURES 16.  17.  18.  19.  PAGE Uro to protoporphyrin (8-2) chromatograms of f e c a l samples SA-a and VGH-la (Table X)  53  Chromatographic i l l u s t r a t i o n of the q u a l i t a t i v e s i m i l a r i t i e s of the f e c a l sub-uro porphyrins; 1.50% methanol i n methylene dichloride analysis  58  Chromatographic i l l u s t r a t i o n of the q u a l i t a t i v e s i m i l a r i t i e s of the f e c a l sub-uro porphyrins; 1.00% methanol i n methylene dichloride analysis  59  I l l u s t r a t i o n of the use of variable wavelength detection and stop-flow v i s i b l e scans for sub-uro porphyrin analysis  61  viii  THIS THESIS WOULD BE INCOMPLETE WITHOUT AN EXPRESSION OF THANKS TO:  Dr. D a v i d D o l p h i n f o r h i s a b i l i t y  to d i f f e r e n t i a t e  Dr. Rozanne P o u l s e n f o r a review o f t h i s  f o r providing c l i n i c a l  from t h e t r e e s .  thesis.  Dr. M e l v i n B e r n s t e i n f o r p r o v i d i n g c l i n i c a l  Dr. Sam Schwartz  the f o r e s t  i n f o r m a t i o n and samples.  samples and h i s f l o r i s i l  procedure.  ix  TO  John and  linda  and most of  all  1  I.  INTRODUCTION  The porphyrias are a group of acquired or inherited metabolic diseases characterized by the abnormal production and excretion of porphyrins and porphyrin precursors.  The c l i n i c a l evaluation and subsequent  treatment of porphyria are dependent on the analysis of the porphyrins and porphyrin precursors present i n urine, feces and blood because a wide array of overlapping p h y s i o l o g i c a l manifestations make d i r e c t  clinical  12 3 diagnosis d i f f i c u l t ' ' . The a n a l y t i c a l techniques currently available are i n e f f i c i e n t and do not have the capacity to separate or e a s i l y quantify complex mixtures 4 5 of porphyrins ' . High pressure l i q u i d chromatography (HPLC), on the other hand,can readily achieve complex separations  and since the sample  being analyzed remains i n s o l u t i o n at a l l times i t s detection t i t a t i o n are considerably s i m p l i f i e d .  and quan-  Bearing these considerations i n  mind, we have coupled new i s o l a t i o n techniques with HPLC and developed rapid and e f f i c i e n t methods f o r the analysis of porphyrins i n urine and feces. A.  CLINICAL AND BIOCHEMICAL ASPECTS OF PORPHYRIA  1.  PORPHYRIN METABOLISM  Porphyrins play important  roles i n many b i o l o g i c a l processes from  oxygen and electron transport to drug d e t o x i f i c a t i o n ^ .  Because of the  v a r i a t i o n i n porphyrin structure and function this introduction considers only those compounds important  to heme biosynthesis and the study of  porphyrin excretion i n porphyria.  2 The patterns of porphyrin excretion i n porphyric patients d i r e c t l y r e f l e c t s the causal abnormalities associated with heme metabolism.  Thus,  an important aspect of the study of porphyrins and porphyria i s to i n crease our understanding  of these processes with a goal of i d e n t i f y i n g  the underlying enzymic defects which are expressed  a.  as porphyric disease.  PORPHYRIN STRUCTURE The porphyrin macrocycle occurs i n heme biosynthesis as a hexa-  hydroporphyrin  (porphyrinogen), porphyrin or porphyrin metal complex"'".  HEXAHYDROPORPHYRIN (PORPHYRINOGEN)  PORPHYRIN  PORPHYRIN METAL COMPLEX  3  Table 1 l i s t s the porphyrin s u b s t i t u t i o n patterns encountered i n t h i s study.  The three most common compounds from t h i s group are:  P  A  UROPORPHYRIN I I I (URO I I I )  P  M  P  P  COPROPORPHYRIN I I I (COPRO I I I )  V  M  P  P  PROTOPORPHYRIN IX (PROTO)  TABLE 1 PORPHYRINS REFERRED TO IN THIS STUDY  UP UP UP UP UP UP UP UP UP UP UP UP UP UP UP UP UP UP  UROPORPHYRIN I I I UROPORPHYRIN I 11EPTACARBOXYIIC ACID PORPHYRIN I I I HEPTACARBOXYLIC ACID PORPHYRIN I HEXACARBOXYLIC ACID PORPHYRIN I I I HEXACARBOXYLIC ACID PORPHYRIN I PENTACARBOXYLIC ACID PORPHYRIN I I I PENTACAREOXYLIC ACID PORPHYRIN I DEIIYDROISOCOPROPORPHYRIN ISOCOPP.OPORPHYRIN DESETHYLISOCOPROPORPHYRIN COPROPORPHYRIN I I I COPROPORPHYRIN I HARDEROPORPHYRIN 2-ETHYL-4-PROPIONIC DEUTEROPORPHYRIN ISOHARDEROPORPHYRIN 2-PROPIONIC-4-ETHYL DEUTEROPORPHYRIN PROTOPORPHYRIN IX HYDROXYETHYLISOCOPROPORPHYRIN HEMATOPORPHYRIN PROTEIN PORPHYRIN ••' c  c  c  gel  POSITIONS  CLASS  PORPHYRIN  UP:  1,8,9,10,11,12  su su su  1  2  3  A  5  6  7  8  A A •A A M M M M M M M M M M M M M M M M M  P P P .P P P P P V E H P P  A A A A A A M M M M M M M M M M M M M M M  P P P P P P P P P P P P P P P V E V P HY R  A A A A A A A A A A A M M M M M M M A M M  P P P P P P P P . P P P P P P P P P P P P P  P A P M P M P M P P P P M P P P P P P P P  A P M P M P M P M M K M P M M H M M M M M  U r o p o r p h y r i n t o p r o t o p o r p h y r i n p o s i t i o n on m e t h y l TLC; SU:  The symbols  silica  silica  g e l TLC.  A - -CH C0 H, P = - C H C H C 0 H , M - - C H , V - -CH=CH , 2  E » - C H C H , HY •= -CHOH.CH 2  ester  S u b - u r o p o r p h y r i n p o s i t i o n on m e t h y l e s t e r  are:  E P P V HY HY R  3  Structure not proved:  2  3>  2  2  2  3  R = -CHS ( P o l y p e p t i d e ) .CH , H - -H. 3  Assumed c o r r e c t by analogy t o t h e I I I system  and u r o I , c o p r o I . d.  A d i v e r s e group  o f p o r p h y r i n s w h i c h may a l s o i n c l u d e monohydroxy-  e t h y l , monopolypeptide'-ethyl p o r p h y r i n s (R(2) o r R ( 4 ) = HY).  2  5  b.  PORPHYRIN BIOSYNTHESIS  5-aminolaevulinic acid  (ALA), t h e f i r s t  compound b i o l o g i c a l l y  committed 0  II  H NCH CCH CH C0 H 2  2  2  2  2  5-Aminolaevulinic to porphyrin biosynthesis, i s derived and «* - o x o g l u t a r a t e .  acid  from a c e t a t e v i a t h e Kreb's  Two m o l e c u l e s o f ALA a r e condensed  t o form t h e monopyrrole  p o r p h o b i l i n o g e n (PBG)'''.  P r  cycle  by ALA d e h y d r a t a s e  Uroporphyrinogen  A  "O M  N' H  kCH NH 2  2  Porphobilinogen  s y n t h e t a s e causes f o u r m o l e c u l e s o f PBG t o condense t o g i v e t h e abnormal u r o p o r p h y r i n isomer:  Uroporphyrinogen  I.  When u r o p o r p h y r i n o g e n s y n -  t h e t a s e a c t s i n the presence of uroporphyrinogen co-synthetase the normal u r o p o r p h y r i n isomer, u r o p o r p h y r i n o g e n I I I , i s formed. d e c a r b o x y l a t i o n of the four carboxymethyl m e t h y l s by u r o p o r p h y r i n o g e n d e c a r b o x y l a s e  groups g  Successive  of uroporphyrin I to  g i v e s the c o r r e s p o n d i n g h e p t a -  c a r b o x y l i c a c i d , h e x a c a r b o x y l i c a c i d , p e n t a c a r b o x y l i c a c i d and c o p r o 1 8 porphyrinogens I . The m e t a b o l i s m  Coproporphyrinogen  I i s not f u r t h e r metabolized ' .  o f u r o p o r p h y r i n o g e n I I I i s diagrammed i n f i g u r e 1.  the p r e s e n c e of normal  and abnormal pathways.  Note  6 UROPORPHYRINOGEN I I I HEPTACARBOXYLIC ACID PORPHYRINOGEN I I I  V  HEXACARBOXYLIC ACID PORPHYRINOGEN I I I PENTACARBOXYLIC ACID PORPHYRINOGEN I I I  Lc DEHYDROISOCOPROPORPHYRINOGEN \ \ \ \  COPROPORPHYRINOGEN I I I  \ \  PROTEIN PORPHYRIN COMPLEXES  \ \ \  ^  \  \  ISOHARDERO PORPHYRINOGEN  HARDEROPORPHYRINOGEN PROTOPORPHYRINOGEN IX PROTOPORPHYRIN IX  v HEME (IRON (II) PROTOPORPHYRIN IX) a-  normal metabolism;  abnormal metabolism  b.  Other intermediates may be present  c.  Observed i n v i t r o  FIGURE 1. UROPORPHYRINOGEN III M E T A B O L I S M ' 3  8,10  ' ' 11  13  7  The  processes  genase and p r o t o g e n  which r e s u l t  i n t h e c o n v e r s i o n by  coproporphyrino-  dehydrogenase^ o f c o p r o p o r p h y r i n o g e n  III v i a proto-  11 13 porphyrinogen  IX t o p r o t o p o r p h y r i n IX a r e not w e l l understood  '  I n p a r t i c u l a r t h e f o r m a t i o n o f a heterogenous group o f c y s t e i n e t h i o e t h e r l i n k e d p r o t e i n p o r p h y r i n i n t e r m e d i a t e s has been observed  i n the i n  11 13 vitro  coproporphyrinogenase r e a c t i o n  '  .  A p o s s i b l e example o f t h i s  group i s d i c y s t e i n y l Nhematoporphyrin IX"'""'". H CH 2  3  H0 CCHCH SCH 2  2  CHCH SCH CHC0 H 3  2  2  |NlH  2  DICYSTEINYL HEMATOPORPHYRIN IX C.  PORPHYRIN EXCRETION  The  compounds o f normal and abnormal p o r p h y r i n metabolism a r e  e x c r e t e d v i a t h e u r i n e and f e c e s as p o r p h y r i n p r e c u r s o r s ,  porphyrinogens,  1 3 porphyrins  and p o r p h y r i n m e t a l complexes  are unstable dation.  they a r e a n a l y z e d  Table I I l i s t s  products  Because the p o r p h y r i n o g e n s  i n the t o t a l p o r p h y r i n f r a c t i o n a f t e r  t h e p o r p h y r i n s w h i c h have been observed  a n a l y s i s o f u r i n e and f e c a l The  ' .  oxi-  i n the  specimens.  o f heme c a t a b o l i s m a r e e x c r e t e d as  p i g m e n t s ^ ' • ^ ' ^ " ' ' ^ and c o n s e q u e n t l y  non-macrocyclic  do n o t p l a y a r o l e i n p o r p h y r i n  analysis.  TABLE II PORPHYRINS FOUND IN PORPHYRIC SAMPLES ' ' ' 1  2  1 0  Uroporphyrin I, I I I Zinc uroporphyrin Heptacarboxylic acid porphyrin I, I I I Hexacarboxylic acid porphyrin I, I I I Pentacarboxylic acid porphyrin I, I I I Dehydroisocoproporphyrin Isocoproporphyrin'  3  Desethylisocoproporphyrin Hydroxyethylisocoproporphyrink Coproporphyrin I, I I I Protein porphyrin complexes . b Hematoporphyrin Harderoporphyrin 2-Ethyl-4-propionic deuteroporphyrin Isoharderoporphyrin 2-Propionic-4-ethyl deuteroporphyrin Protoporphyrin IX  Unknown isomer type Probably formed by gut microflora  1 : L  9  2.  CLASSIFICATION OF THE PORPHYRIAS:  CLINICAL AND BIOCHEMICAL SYMPTOMS  At least nine d i s t i n c t porphyric diseases are recognized III)  (Table  1,3 with p h y s i c a l symptoms spanning the range from minimal discomfort  to severe  mental i l l n e s s , p a r a l y s i s and death. TABLE I I I CLASSIFICATION OF THE PORPHYRIAS Erythropoietic  porphyria  Congenital e r y t h r o p o i e t i c porphyria congenita,  porphyria  Erythrohepatic protoporphyria erythropoietic  (EHPP), f o r m e r l y  protoporphyria  Erythrohepatic coproporphyria  (EHCP), f o r m e r l y  e r y t h r o p o i e t i c coproporphyria, hepatic  Hereditary  porphyria  Gunther's d i s e a s e  Erythrohepatic  The  (CEP),  coproporphyrinemia  porphyrias forms  Acute intermittent porphyria porphyria, pyrrolia,  (AIP), Swedish g e n e t i c  pyrroloporphyria  Hereditary coproporphyria  (HC), i d i o p a t h i c  copropor-  phyrinuria Variegate porphyria  (VP), South A f r i c a n  genetic  p o r p h y r i a , mixed p o r p h y r i a , p o r p h y r i a  cutanea  tarda hereditaria, Symptomatic forms:  protocoproporphyria  symptomatic p o r p h y r i a  (SP),  p h y r i a c u t a n e a t a r d a symptomatica, a c q u i r e d secondary  p o r p h y r i a , Bantu p o r p h y r i a ,  porphyria,  por-  porphyria,  constitutional  urocoproporphyria  SP a s s o c i a t e d w i t h a l c o h o l abuse (SP-A) p l u s  iron  overload SP  induced  by c h e m i c a l s  (SP-C), hexachlorobenzene  (HCB), p e n t a c h l o r o p h e n o l , chemical fungal  metabolites  SP a s s o c i a t e d w i t h d i s e a s e and  s t e r o i d drugs and o t h e r  compounds, and p o s s i b l y b a c t e r i a l and  a miscellaneous  (SP-D), h e p a t i c  porphyrinoma,  group o f r a r e d i s e a s e s .  10  Unfortunately, the heterogeneity of c l i n i c a l responses (Tables IV and V) which r e s u l t from the variety as well as the c y c l i c a l (acute  3 attack:remission)  ;  nature of some of the porphyrias  diagnosis and subsequent treatment d i f f i c u l t .  make correct  In addition, the secondary  3 coproporphyrinurias are non-porphyria  diseases  resulting i n abnormal  coproporphyrin excretion which further complicates  interpretation.  TABLE IV MAIN CLINICAL FEATURES OF THE PORPHYRIAS  Type of Porphyria  Usual Age of Onset (years)  Mode of Inheritance  Mechanical Dermatosis.  Acute Photosensitivity  Acute Attacks  +  0  Erythropoietic CEP  Congenital; Recessive  Erythrohepatic EHPP  Under 10  Dominant  +  0  EHCP Hepatic  Under 10  Dominant  +  0  AIP HC VP SP-A  Under 30 Under 10 Under 30 Over 40  Dominant Dominant Dominant 0  0 + ' + +  0 (+) (+) (+)  + + + 0  SP-C  Any age  0  +  (+)  0  SP-D  Over 60  0  +  (+)  0  Key:  +  + = present,(+) = very r a r e l y present, 0 = absent.  11  TABLE V CLINICAL FEATURES OF THE ACUTE ATTACK: A COMPARISON OF PERCENTAGE INCIDENCE 3  Sweden [233] (%) a  U.K. [50] (%)  U.S.A. [69] (%)  S. A f r i c a [80] (%)  Mai es  40  38  39  30  Females  60  62  61  70  Abdominal pain  85  94  95  90  Vomiting  59  78  52  80  Mental changes^  65  74  80  67  Constipation  48  74  46  80  Paralysis  42  68  72  53  Hypertension  40  56  49  55  Pyrexia^  37  14  36  38  Tachycardia  28  64  51  83  Sensory loss  9  38  24  15  Diarrhea  9  12  11  8  Azotemia^  9  1  67  69  Proteinuria®  9  14  —  7  24  48  20  Cranial nerves  1  29  51  9  ECG abnormalities  1  44  47  23  C  Leukocytosis'  1  8  a.  Bracketed figures refer to the number of patients i n each s e r i e s .  b.  May include delirium, hysteria, apathy and epileptiform  c.  E s p e c i a l l y respiratory p a r a l y s i s .  d.  Fever  e.  Abnormally rapid heart action.  f.  An excess of nitrogenous bodies i n the blood as a result of kidney insufficiency.  g.  The presence of protein i n the urine.  h.  Increase i n the number of blood leukocytes.  seizures.  12  Therefore, a d d i t i o n a l information from biochemical a n a l y s i s i s necessary f o r d i a g n o s t i c d i f f e r e n t i a t i o n .  Table VI gives a composite of  the normal and abnormal porphyrin and porphyrin precursor patterns which aid i n the c o r r e c t diagnosis of porphyria.  Figure 2 i n d i c a t e s the regions  of the heme b i o s y n t h e t i c pathway believed to contain the malfunctioning metabolic enzyme(s).  This o u t l i n e i s the r e s u l t of the study of porphyrin  13 excretion patterns  . A d d i t i o n a l research i s necessary to i d e n t i f y the  exact malfunction responsible f o r each disease.  T  ALA  HEREDITARY COPROPORPHYRIA  ACUTE INTERMITTENT PORPHYRIA UROPORPHYRINOGEN III  1  VARIEGATE PORPHYRIA  UROPORPHYRINOGEN I  SYMPTOMATIC PORPHYRIA  CONGENITAL PORPHYRIA COPROPORPHYRINOGEN III . COPROPORPHYRINOGEN I  PROTOPORPHYRIN IX  FIGURE 2.  T  ERYTHROHEPATIC COPROPORPHYRIA  ERYTHROHEPATIC PROTOPORPHYRIA  REGIONS OF THE HEME BIOSYNTHETIC PATHWAY AFFECTED IN THE PORPHYRIAS ' . 1  17  13  TABLE VI  VALUES FOR URINARY, FECAL AND ERYTHROCYTE PORPHYRINS AND PORPHYRIN PRECURSORS IN PORPHYRIC AND NORMAL P A T I E N T S  1,3  Type o f Porphyria  Blood  Urine  Normal  P r o t o 4-52 ug/100 ml Copro 0-4 ug/100 ml  ALA 2.5 mg/day P r o t o 0-76 ug/g PBG 10 mg/day Copro 0-20 ug/g C0PR0 0-160 y g / l i t e r E t h e r - I n s o l u b l e URO 6-30 y g / l i t e r P o r p h y r i n 0-22  Feces  Isomer type  Vg/g  III  c  Congenital Erythropoietic  Increased (mainly uro)  ALA, PBG - normal URO-much i n c r e a s e d COPRO-increased  PROTO-some i n c r e a s e COPRO-increased URO-some i n c r e a s e SUB-URO-not d e t e r mined  Erythrohepatic Protoporphyria  Increased (mainly proto)  U s u a l l y normal  PROTO-increased COPRO-sometimes increased  III  Erythrohepatic Coproporphyria  Increased (mainly copro)  U s u a l l y normal  U s u a l l y normal  III  Acute I n t e r m i t t e n t Porphyria  Normal  ALA.PBG-increased URO-may be p r e s e n t ^  Normal  H e r e d i t a r y Coproporphyria  Normal  ALA,PEG-may be i n creased COPRO-increased  COPRO-increased SUB-URO-slight increase  Variegate Porphyria  Normal  ALA,PEG-increased COPRO,URO-may be increased with COPRO g r e a t e r t h a n URO  PROTO,COPRO-large M a i n l y I I I I n c r e a s e w i t h PROTO g r e a t e r t h a n COPRO SUB-URO-large i n crease  Symptomatic phyria  Normal  ALA.PBG-normal URO-large i n c r e a s e  Por-  a.  ug/100 ml o f packed  b.  Ug/g o f d r y s t o o l  c.  T h i s f r a c t i o n may i n c l u d e h e p t a c a r b o x y l i c a c i d ,  d.  Uro may be p r e s e n t  e.  May be normal d u r i n g  e  cells  u r o and s u b - u r o  from t h e non-enzymatic c o n d e n s a t i o n remission  o f PBG  porphyrins  III  PROTO,COPRO-moderComplex ate increase with COPRO g r e a t e r than PROTO ETHER-INSOLUBLE-increased  14  B.  CURRENT ANALYTICAL METHODS FOR THE DETERMINATION OF PORPHYRINS AND PORPHYRIN PRECURSORS)IN CLINICAL SAMPLES  The currently employed a n a l y t i c a l methods can be divided into q u a l i t a t i v e methods which are useful for screening and preliminary diagnosis and quantitative methods which are a necessity f o r the p o s i t i v e i d e n t i f i c a t i o n of the s p e c i f i c porphyria. 1.  URINE SAMPLES  a.  PORPHYRIN PRECURSORS The porphyrin precursors ALA and PBG are quantitated by formation of  a red-coloured complex with Ehrlich's reagent benzaldehyde i n 5 N. HC1)  (2% w/v  p-dimethylamino-  after ion-exchange preparation of the urine  -, 3,18 sample  b.  URO  TO PROTO PORPHYRINS  Urine samples are screened for abnormal porphyrin concentration by spectrophotometry  a f t e r q u a l i t a t i v e porphyrin separation by a c i d i f i c a t i o n  and extraction with ether"*.  This procedure y i e l d s ether soluble (mostly  coproporphyrin) and ether insoluble (mostly heptacarboxylic acid porphyrin and uroporphyrin)  fractions.  The quantitative analysis of a sample i s much more complex as 19 outlined by the procedure of Dowdle The pH of the urine sample was adjusted to between 3.0 and 4.0 with g l a c i a l acetic adid; and approximately l/10th volume of powdered t a l c was added. The s l u r r y was s t i r r e d for lhn,after which the t a l c and adsorbed porphyrins were harvested by f i l t r a t i o n with suction. This procedure was repeated u n t i l an acetic acid:ether:amyl acohol (1:1:1) extract of the f i l t r a t e no longer fluoresced under u l t r a - v i o l e t l i g h t . The t a l c was then washed with 1% (v/v) aqueous acetic acid and water and thoroughly dried i n a vacuum dessicator over s i l i c a g e l .  15  •  The adsorbed p o r p h y r i n s were e l u t e d and e s t e r i f i e d i n one s t e p by suspending the t a l c i n methanol: H2SO4 (20:1) and a l l o w i n g the suspension to stand o v e r n i g h t i n the dark. The suspension was then f i l t e r e d and the t a l c washed w i t h methanol: H2SO4 (40:1) u n t i l i t no l o n g e r f l u o r e s c e d . The pooled f i l t r a t e and washing were a d j u s t e d to pH 4.0 w i t h s a t u r a t e d sodium a c e t a t e and the p o r p h y r i n methyl e s t e r s were e x t r a c t e d i n t o c h l o r o f o r m . The c h l o r o f o r m s o l u t i o n -ii of methyl e s t e r s was washed f o u r times w i t h d i s t i l l e d water, and taken to dryness under reduced p r e s s u r e .  The (TLC) and  e x t r a c t e d p o r p h y r i n s a r e separated by t h i n l a y e r chromatography q u a n t i t a t e d i n s i t u w i t h a f l u o r e s c e n c e scanner or 2  spectrophoto-  20  m e t r i c a l l y a f t e r e l u t i o n from t h e p l a t e '  .  See f i g u r e 3b f o r a  r e p r e s e n t a t i v e f l u o r e s c e n c e scan of a p o r p h y r i n sample separated The  isomeric composition  by the h y d r o l y s i s and acid, hexacarboxylic  The  TLC.  of a sample (I and/or I I I ) i s determined 21  decarboxylation  of the i s o l a t e d uro,  heptacarboxylic  a c i d o r p e n t a c a r b o x y l i c a c i d p o r p h y r i n to  p h y r i n f o l l o w e d by o v e r n i g h t chromatography u s i n g a system.  by  decarboxylation i s necessary  copropor-  paper/lutidene  because the r e s p e c t i v e i s o m e r i c  uro to p e n t a c a r b o x y l i c a c i d p o r p h y r i n s have not been s u c c e s s f u l l y separated 22 by chromatography 2.  FECAL SAMPLES  a.  URO  TO PROTO PORPHYRINS  The  q u a l i t a t i v e s c r e e n i n g of f e c a l samples a l s o uses an a c i d / e t h e r  p a r t i t i o n f o l l o w e d by a f u r t h e r s e l e c t i v e e x t r a c t i o n f o r s e p a r a t i o n of p r o t o p o r p h y r i n and  coproporphyrin  fractions^.  Although  t h i s procedure  g i v e s u s e f u l g e n e r a l i n f o r m a t i o n the a n a l y s i s p r o v i d e s l i t t l e or no i n f o r m a t i o n on the more p o l a r p o r p h y r i n s  (uro, h e p t a c a r b o x y l i c a c i d ) and  the  16  a p p l i c a t i o n of TLC to the i s o l a t e d f r a c t i o n s  4  (Figure 3a) after  methanol/R^SO^ e s t e r i f i c a t i o n i l l u s t r a t e s the lack of porphyrin r e s o l u t i o n Quantitative  analysis i s s i m i l a r to the TLC method used for urine  a f t e r extraction of the f e c a l porphyrins as t h e i r methyl esters with  4 methanol/H^SO^ of methanol/BF^ . Figure 3b i l l u s t r a t e s the a n a l y t i c a l r e s o l u t i o n normally obtained with t h i s procedure. quantitative separation  I f the analysis  requires  and i s o l a t i o n of s p e c i f i c porphyrin components i n  the sample a d d i t i o n a l extraction and/or chromatographic steps are usually 9 20 23 24 25 necessary which greatly add to the time and complexity of analysis '  '  Isomer a n a l y s i s i s the same as f o r urine samples.  © u  Proto  ! ft Copro  FIGURE 3a.  TLC ANALYSIS OF ACID/ETHER EXTRACTED PROTO AND COPRO FRACTIONS FROM PORPHYRIC FECES . 4  8-2 - Uro to proto porphyrins.  ' .  17  FIGURE 3b.  TLC/CHROMATOSCAN CHROMATOGRAM OF A PORPHYRIC SAMPLE 8-2 - Uro to proto porphyrins;  .  SU - Sub-uro porphyrins.  18  b.  SUB-URO PORPHYRINS  The sub-uro porphyrins are determined  q u a l i t a t i v e l y by observation of 20 26  red-fluorescent bands i n the sub-uro area of the developed TLC plate or semiquantitatively using the extraction technique of Rimington "'" 1  outlined here. Ether soluble porphyrin (copro, proto) was removed by s i x successive extractions with ether-acetic acid (10/1). The residue was l e f t overnight i n the urea-Triton mixture (45% (w/v) urea containing 4% (v/v) T r i t o n X-100) and then c e n t r i fuged. Extraction was t h r i c e repeated and the combined extracts shaken twice with n-butanol. After washing t h i s solvent; 1 volume of ether and 0.5 volume l i g h t petroleum was added and the porphyrin extracted by repeated shaking with small amounts of 5% HC1. The aqueous acid extract so obtained was suitable for spectrophotometric quantitation but s t i l l contained amounts of T r i t o n which interfered with p u r i f i c a t i o n procedures. Its removal presented a d i f f i c u l t problem, solved eventually by adsorbing the porphyrin material onto calcium hydroxide (pH 8), centrifuging and then washing the s o l i d phase exhaustively with 50% (v/v) aqueous ethanol. This removed a l l the detergent and the adsorbant could then be r e d i s solved by addition of a s l i g h t excess of d i l u t e HC1. Usually, most of the porphyrin-containing material dissolved r e a d i l y i n the acid but with some preparations, especially those from faeces, acid-soluble and acid-insoluble fractions were obtained.  Although some of the sub-uro compounds have been i d e n t i f i e d as thio-ether linked protein porphyrins of molecular weight 1000 to 6000"*" 26 or as hydroxyethylisocoproporphyrin components by TLC  the lack of resolution of these  (Figure 3a) and the extra processing steps necessary  '  19  to remove ^contaminating T r i t o n X-100  from the Rimington samples before  further separation has severely hindered the development of routine analytical  3.  procedures.  BLOOD SAMPLES  The a n a l y t i c a l procedure for blood sample free protoporphyrins uses the outlined lengthy and tedious set of extraction steps to remove 27 heme and other impurities Quantitative Determination of Free Erythrocyte Protoporphyrin  1. Measure 5 ml of heparinized blood into a 50 ml conical centrifuge tube, centrifuge, and remove the plasma. 2. Mix the c e l l s thoroughly with 25 ml of ethyl acetate/ acetic-acid - adding the solvent slowly with constant stirring. 3. Allow the mixture to stand u n t i l the f i n e reddishbrown p r e c i p i t a t e s e t t l e s to the bottom. 4. Decant the supernatant through f i l t e r paper into a 250 ml separatory funnel. 5. Wash and s t i r the p r e c i p i t a t e with three successive 15 ml portions of the extraction mixture allowing the prec i p i t a t e to s e t t l e out between each wash and decanting the supernatant into the same separatory funnel through the same f i l t e r paper after each extraction. *6. Wash the f i l t r a t e with three successive 20 ml portions of H2O c o l l e c t i n g the washings i n a second 250 ml separatory funnel. (Allow at least 10 minutes f o r complete separation of the aqueous and organic phases after the f i n a l washing at the points indicated by an asterisk i n the method.) *7. Back extract the water washings with 15 ml ethyl acetate to remove any porphyrins which may have been washed out. Add the ethyl acetate to the ethyl acetate, acetic acid extraction mixture and discard the aqueous layer. 8. Extract the porphyrin from the ethyl acetate with successive 5 ml portions of 10% HC1 u n t i l no red f l u o r e scence can be detected i n the acid layer under u l t r a v i o l e t l i g h t . A minimum of two extractions i s required.  20  9. Add saturated sodium acetate to the collected HC1 ext r a c t s u n t i l they are basic. 10. Add 2 ml of g l a c i a l acetic acid to the extracts to reduce the tendency to form emulsions. 11. Extract the HC1 solution with successive 25 ml portions of ethyl ether u n t i l no red fluorescence can be seen i n the ether extraction under u u l t r a v i o l e t l i g h t . Normally two extractions are s u f f i c i e n t . Collect the ether extractS'-in a separatory funnel. 12. Wash the ether extracts with 5 ml of 1% Na2C03. D i s card the aqueous layer. *13. Wash the ether extracts twice with 5 ml portions of d i s t i l l e d H2O. *14. Extract the ether with 1.5 ml of 25% HC1. Allow a very good separation of the phases and extract a second time with approximately 0.5 ml of 25% HC1. Collect the f i r s t extract i n a graduated centrifuge tube. 15. Check the second extract with an u l t r a v i o l e t l i g h t to ensure that a l l fluorescence has been removed before adding i t to the f i r s t extract. In normal or s l i g h t l y elevated protoporphyrin levels a l l fluorescing substances w i l l be removed i n the f i r s t 25% HC1 extraction. I f fluorescence p e r s i s t s , continue to extract with 0.5 to 1.0 ml quantities of 25% HC1 u n t i l the f l u o r e scent material i s completely removed. 16. Measure the volume of the HC1 extracts. 17. Read the OD at the peak of the Soret band (407 mu) and at 430 my and 380 mu. Calculate the protoporphyrin concentration. If Congenital erythropoietic porphyria or Erythroheptic coproporphyria are suspect and the composition of the blood porphyrins i s des i r e d step 5 of the above procedure i s followed by e s t e r i f i c a t i o n and 2 chromatography . Occasionally, t h i s procedure i s augmented with aqueous HC1 extraction f o r the t o t a l quantitation of uroporphyrin^.  21  C.  HPLC BACKGROUND  1.  RESOLUTION THEORY Resolution i s a measure of the degree of separation of a system  and i s the c r i t e r i a used to determine the success of a s p e c i f i c graphic procedure  28  .  I t i s dependent on  chromato-  28 29 ' :  S e l e c t i v i t y - a measure of r e l a t i v e compound retention Capacity - a measure of analysis time defined as k' = volume of sample e l u t i o n (Ve) - void volume of column (Vo)/Vo Random dispersion - a measure of the e f f i c i e n c y of a column (peak band width) which i s defined i n terms of the height equivalent of the t h e o r e t i c a l plate (H) such that H = A + Cu . 11  The t h e o r e t i c a l plate i s  defined as the column volume element of minimum dimension such that the p a r t i t i o n i n g of the solute between the mobile and stationary phases reaches equilibrium before moving on:to the next volume element.  A is a  measure of eddy d i f f u s i o n or non-homogeneous flow and i s dependent on p a r t i c l e size.  Cy  11  i s a measure of the non-equilibrium which r e s u l t s  from resistance to mass transfer i n the stationary and mobile phases.  The  smaller the value of H the more plates/meter generated and the greater the e f f i c i e n c y of resolution. i n l i q u i d chromatography  This i s usually the most important factor  and i s the cause of the large e f f e c t on both  stationary and mobile phase mass transfer seen with p a r t i c l e size reduction.  A d d i t i o n a l l y , H may be decreased by peak assymmetry which r e s u l t s  from the effect of multiple mode mass transfer (co-occurance of several d i f f e r e n t adsorption/desorption processes).  22  The increased resolution which r e s u l t s from decreased p a r t i c l e size was  the p r i n c i p l e factor which lead to the development of high pressure 28  (speed) l i q u i d chromatography  .  As the average p a r t i c l e size decreases  analysis time increases unless the solvent i s pumped through the column. Modern HPLC systems are capable of 10 ml/min at 6000 - 8000 PSI. 2.  SEPARATION MODES  Liquid chromatography uses adsorption, reverse phase adsorption, ion-exchange or permeation modes of separation. Adsorption, reverse phase adsorption and ion-exchange operate by v a r i a t i o n of the forces a t t r a c t i n g solutes to the adsorbent tending to remove them from the adsorbent b i l e phase.  and the forces  so they w i l l move with the  mo-  Adsorption chromatography uses a non-polar moving phase  and a polar stationary phase while reverse phase chromatography uses a 28 polar moving phase and a non-polar  stationary phase  Permeation chromatography i s based not on adsorption/desorption processes but on solute molecular size separations by d i f f u s i o n through a r i g i d gel notework.  The larger solute w i l l be more d i f f u s i o n  restric-  ted and w i l l elute before smaller molecule solutes.  Frequently, however, 28 adsorption processes occur with permeation processes i n a gel separation 3.  PORPHYRIN HPLC  The a p p l i c a t i o n of HPLC to the analysis of ; n a t u r a l l y occurring porphyrins was unknown before this work was  initiated  30  .  Subsequently, i n  23  addition to the procedures reported here  '  , techniques have been  17 33 developed f o r the analysis of uro to proto porphyrins of harderoporphyrin/isoharderoporphyrin  '  and the separation  and the isomeric coproporphyrins  .  II.  EXPERIMENTAL  Uv-vis spectra were recorded on a Cary 17 spectrophotometer.  Mass  spectra were recorded on a Varian/mat CH4B using d i r e c t probe i n s e r t i o n at 70 eV and 230° - 330°.  The HPLC was a Waters Assoc. ALC 202.  A.  HPLC INSTRUMENTATION  1.  THE HIGH PRESSURE LIQUID CHROMATOGRAPH.  Figure 4 i s a diagrammatic representation of the components of the HPLC system used f o r these studies. Solvent programs were generated by changing the solvent  reservoir  or by displacement of solvent from a 12.5 ml loop (3.8 meters of 2 mm ID s t a i n l e s s s t e e l tubing) placed on the valve and loop i n j e c t o r . SOLVENT  PUMP  VALVE/LOOP INJECTOR SEPTUM INJECTOR COLUMN 254 nm DETECTOR  FLOW CARY 17 DETECTOR  COLLECTION/WASTE  FIGURE 4.  SCHEMATIC OF AN HPLC SYSTEM  25  2.  SPECIAL APPARATUS:  A VARIABLE WAVELENGTH AND SCANNING DETECTOR  Figure 5 i l l u s t r a t e s the design of a flow c e l l f o r a Cary 17 spectrophotometer.  This c e l l allows detection i n the uv - v i s i b l e and stop-  flow uv - v i s i b l e scans.  FIGURE 5.  The sample volume i s ca. 60 u l .  DIAGRAM OF THE HPLC/CARY 17 FLOW CELL. The quartz right-hand dimensions 2 x 7.4 mm  windows one has are 2.5 (quartz  are symmetrically disposed, only the been sectioned i n the drawing. The body x 2.5 cm and the solvent beam path i s to quartz).  26 i  B.  SOLVENT AND COLUMN PREPARATION  1.  SOLVENTS  Methanol and a c e t o n i t r i l e were spectro quality. ide,  Ammonium hydrox-  n-propanol, triethylamine (TEA), ethyl acetate, ethyl propanoate  and propyl acetate were reagent grade.  Propyl propanoate was made by  s t i r r i n g propionic acid i n propanol/H^SO^ overnight followed by extraction and d i s t i l l a t i o n .  The l i g h t petroleum (30-60°) and methylene dichloride  were glass d i s t i l l e d and had 10 u l TEA/100 ml added unless otherwise indicated.  The glass d i s t i l l e d methylene dichloride/TEA solution was  used within 5 days of d i s t i l l a t i o n .  Older solutions began to exhibit  altered chromatographic properties. Solvent combinations were made on a v/v basis.  A l l columns were  flushed with an excess of the trace solvents before e q u i l i b r a t i o n with the desired, solvent mixture.  2.  COLUMNS  Polyamide, C o r a s i l C-18, Neutral alumina, Basic alumina, P o r a s i l C, P o r a s i l T and C o r a s i l II (Table VII) were dry packed i n stainless s t e e l chromatography  columns.  was s l u r r y packed with methanol/methylene  2.0 mm x 60 cm  Sephadex LH-20 (Table VII) dichloride (1/1) i n a 7.5 mm  x 120 cm stainless steel.chromatography column.  27 TABLE VII DESCRIPTION OF THE HPLC COLUMN PACKINGS USED IN THIS STUDY  PACKING  SOURCE  DESCRIPTION  Polyamide  A iiylon type adsorbent bonded on an impermeable bead  Reeve Angel  C o r a s i l C-18  A linear 18-carbon alkane bonded to a s i l i c a coating on an impermeable bead. P a r t i c l e size 37-50 ym.  Waters Assoc.  Neutral Alumina  A neutral alumina with p a r t i c l e s i z e 18-30 um.  Waters Assoc.  Basic Alumina  A basic alumina with p a r t i c l e 18-30 urn.'  Waters Assoc.  Porasil C  A spherical t o t a l l y porous s i l i c a with p a r t i c l e size 37-75 ym.  Waters  Porasil  An irregular t o t a l l y porous s i l i c a with p a r t i c l e size 25-37 um.  Waters Assoc.  C o r a s i l II  A s i l i c a coating on an impermeable bead with p a r t i c l e size 37-50 ym.  Waters Assoc.  Sephadex LH-20  A semi-rigid bead formed dextran g e l .  Pharmacia  T  size  Assoc,  28 C.  PORPHYRIN IDENTIFICATION AND QUANTITATION PROCEDURES  1.  PORPHYRIN STANDARDS The following samples were used as standards:  Uroporphyrin I I I  from copper uro III (Porphyrin products) demetallated with ^SO^ and e s t e r i f i e d with BF^/methanol; Uroporphyrin I (Porphyrin products) e s t e r i f i e d with diazomethane; coproporphyrin I and I I I were samples found i n our lab; protoporphyrin IX (prepared by RK DiNello) e s t e r i f i e d with BF^/ methanol.  The hepta-, hexa- and penta- carboxylic acid porphyrins were 21  obtained by sealed tube degradation of uroporphyrin methane e s t e r i f i c a t i o n . porphyric urine samples.  followed by diazo-  The t r i c a r b o x y l i c acid porphyrin was isolated from Copper protoporphyrin IX and copper 36  phyrin I I I were made by the procedure of Doss  .  copropor-  Hematoporphyrin  (Nutritional biochemicals) was e s t e r i f i e d with BF^/methanol. propyl esters were prepared by ethanol or propanol/BF^  Ethyl and  esterification  of the r e s p e c t i v e porphyrins. 2. STOP-FLOW VISIBLE SPECTRA V i s i b l e spectra of compounds eluted from the HPLC column were made by stopping the solvent flow when the sample was i n the Cary 17 flow c e l l and scanning the desired wavelength range. 3.  MASS SPECTRA  Samples of HPLC eluted porphyrins were collected (ca 1 yg) and 37 transferred to a crucible f o r d i r e c t probe i n s e r t i o n  29  4.  QUANTITATION  Standard samples of uroporphyrin and protoporphyrin were used to c a l i b r a t e microgram porphyrin/peak area response. detection system at 403.5 nm,  Using the Cary 17  .2 absorbance units f u l l scale and 10.4  min. chart speed a value of .0192  mm/  ± .005 ug/area unit (ca ±3%) with a  minimum detection response of ca 25 picogram/peak was obtained.  D.  PRE-ANALYSIS PREPARATION OF PORPHYRIC SAMPLES  1.  URINE SAMPLES  Solid sodium bicarbonate (0.1-0.3%) was added to the urine sample and the solution was allowed to stand at room temperature overnight. Florisil  (1 g, 100-200 mesh) was suspended i n 10% hydrochloric acid (25  ml) and poured into a glass chromatography column (1.2 cm diameter). When the f l o r i s i l had s e t t l e d , .celite (300 mg;  the c e l i t e was previously  washed with 20% HC1 to remove a yellow pigment) i n d i s t i l l e d water (25 ml) was added to the column.  A 10 ml urine sample was then c a r e f u l l y  a c i d i f i e d with 25% hydrochloric acid (one f i f t h the volume of urine) and after a b r i e f degassing on a water aspirator the sample was added to the column. HC1,  The column was washed with successive 25-ml aliquots of 5%  0.1% HC1,  10% acetic acid: 95% ethanol (3:1; an additional volume  of t h i s solution was needed occasionally to completely elute a yellow 38 fluorescing compound) and d i s t i l l e d water  .  The porphyrins were eluted  with a minimum volume of 3% T r i s (containing 2 ppm disodium EDTA): 95% ethanol (2:1).  30  The porphyrin-containing eluate was diluted to twice i t s volume with 95% ethanol.  This s o l u t i o n was cooled to 0°C and reacted with an ether39  eal solution of diazomethane  (3-6 ml, 10 mg/ml).  Ether (20 ml) and  water (20 ml) were added, and after shaking the aqueous layer was d i s carded.  The ether layer was washed twice with 20 ml of d i s t i l l e d water.  The ether was then removed on a rotary evaporator.  Methylene dichloride  (10 ml) was added to the residue and the organic phase dried over anhydrous sodium sulphate.  The mixture was f i l t e r e d and the f i l t r a t e  to dryness on a rotary evaporator.  taken  The residue was dissolved i n methylene  d i c h l o r i d e (0.50 ml) f o r analysis. 2.  FECAL SAMPLES  20 ml of methanol and 2 ml of BF^'Et20 were added to the f e c a l  4 sample (.5 g wet or .2 g dry) .  The solution was s t i r r e d overnight.  The e s t e r i f i e d material was extracted with two 100 ml portions of methylene dichloride after the addition of 80 ml of water.  The methylene  d i c h l o r i d e solution was dried over anhydrous sodium sulphate, f i l t e r e d and the solvent removed on a rotary evaporator. A sample of the residue was dissolved i n methylene d i c h l o r i d e (1.0 ml/.10 g dry feces) and analysed by HPLC gel chromatography.  The residue  was dissolved i n a minimum of solvent and chromatographed on 60 g of s i l i c a gel Woelm a c t i v i y IV i n a 2.5 x 45 cm glass column. was benzene/ethyl  acetate/methanol  (40/10/2).  The solvent  A yellow-brown material  eluted f i r s t followed by the uro to proto porphyrins and f i n a l l y by a  31  sub-uro porphyrin band.  A"typical column gave:  (10 ml fractions collected  commencing when the sample was added to the column) Fractions Uro to proto porphyrins  6-26  Sub-uro porphyrins  28-35  After the appropriate fractions were pooled the solvent was removed on a rotary evaporator and the residue dissolved i n methylene dichloride (1.0 ml/.10 g dry feces) f o r analysis. In addition to the standard f e c a l sample preparation procedure described above a large sample of porphyric feces (VGH - l b ) which contained s i g n i f i c a n t quantities of sub-uro material was prepared f o r analysis. 125 ml of methanol and 8 ml of B F ' E t 0 were added to a 1.254 g 3  (dry weight) f e c a l sample.  2  The solution was s t i r r e d overnight and extrac-  ted with 3 x 500 ml portions of methylene dichloride as described previously.:. A f t e r measurement of the t o t a l Soret absorption the residue was dissolved i n a minimum of solvent (as above) and chromatographed on 350 g of s i l i c a g e l Woelm a c t i v i t y IV i n a 5.6 x 60 cm glass column. fractions were:  Discard Uro to proto porphyrins Sub-uro porphyrins  400 ml 1055 ml 900 ml  The  32  After the fractions were measured for t o t a l Soret absorption the suburo residue was dissolved i n a minimum of solvent and chromatographed on 180 g of s i l i c a gel Woelm a c t i v i t y IV i n a 2.3 x 60 cm glass column. The solvent was benzene/ethyl  acetate/methanol  (30/20/2).  The fractions  were:  Discard  265 ml  Sub-uro A  250 ml  Sub-uro B  120 ml  After the solvent was removed on a rotary evaporator the samples were weighed and measured for t o t a l Soret absorption.  E.  SPECTROSCOPIC ANALYSIS OF THE SUB-URO FRACTION 100 u l of the sub-uro f r a c t i o n obtained by column chromatography  was diluted to 2.5 ml with methylene d i c h l o r i d e and the absorption measured at 380 nm, 460 nm and the Soret maximum.  The Soret absorption  was corrected for impurities using the formula: A(corrected) = 2A(Soret) - (A(460) + A(380)) 1.48 40 41 This formula i s s i m i l a r to those developed  by Rimington  '  , with the  value 1.48 derived from a standard sample of protoporphyrin IX dimethyl ester. Selected-.fecal samples were also analyzed for sub-uro porphyrins by the u r e a - T r i t i o n extraction procedure of Rimington . 11  33  F.  HPLC OF CLINICAL SAMPLES  1.  URINE SAMPLES  Urine samples were analyzed on a P o r a s i l T column using a change i n solvent reservoir from l i g h t petroleum d i c h l o r i d e (TEA)/n-propanol  (175/100/1) to l i g h t petroleum  TEA)/methylene dichloride (TEA)/n-propanol sample i n j e c t i o n .  (30-60°; no TEA)/methylene (30-60°; no  (40/100/1) at 14 ml after  The flow rate was 1.5 ml/min.  2.  FECAL SAMPLES  a.  URO TO PROTO PORPHYRINS  The uro to proto porphyrin f r a c t i o n was analyzed on a C o r a s i l I I column using the valve and loop injector to introduce a l i g h t  petroleum  (30-60°;TEA)/methylene d i c h l o r i d e (TEA) (40/100) solvent into a system pumping a l i g h t petroleum n-propanol  (30-60°;TEA)/methylene dichloride (TEA)/  (15/100/.5) solvent.  The flow rate was 1.0 ml/min and the  sample was inj.ected 2.5 min after solvent " i n j e c t i o n " .  b.  SUB-URO PORPHYRINS  The sub-uro porphyrin f r a c t i o n was analyzed on a C o r a s i l II column using methylene dichloride/methanol (98.5/1.5; 99/1) solutions and a flow rate of 1.0 ml/min.  34  G.  IDENTIFICATION OF THE "TRICARBOXYLIC ACID PORPHYRINS"  1.  ANALOGUES OF HARDEROPORPHYRIN  Separation by thin layer chromatography ethyl acetate/methanol (80/2/1):  (TLC) (methylene d i c h l o r i d e /  Rf; proto (.87), t r i c a r b o x y l i c acid  porphyrin (.67), copro (.48)):of the red fluorescent  band i n the t r i c a r -  boxylic acid porphyrin position from VGH - 1 gave compound(s) with an aetio-type spectrum i n methylene  dichloride.  Peak (nm)  Relative absorption  398 501 533 564.5 622  19.7 1.00 .73 .66 .20  The mass spectrum of t h i s i s o l a t e had major peaks at m/e 650 and 652.  2.  COPPER COPROPORPHYRIN  The TLC's i n t h i s section were developed with solvent A (methylene dichloride/ethyl acetate/methanol (40/1/1)) or solvent B (methylene dichloride/methanol/formic acid  (400/3/3)).  The stop-flow v i s i b l e scan of the compound i n the t r i c a r b o x y l i c acid porphyrin p o s i t i o n of the HPLC analysis of f e c a l samples VGH - 2 gave a metalloporphyrin spectrum.  35  Relative absorption  Peak (nm)  27.8 1.00 1.72  399 525 561.5 A sample of t h i s compound  was obtained by TLC (solvent A) c o l l e c t i o n  of the pink,nan^fluorescent band at Rf = .71. metallated with H„S0, but not with HC1. I 4  This compound was de-  Using solvent A TLC the metal  free porphyrin had the same Rf (.55) as an authentic sample of coproporphyria  A synthetic sample of copper coproporphyrin had the same Rf i n  solvent A and the same v i s i b l e spectrum i n methylene d i c h l o r i d e as the unknown.  Confirmation of the unknown as copper coproporphyrin was pro-  vided by i t s mass spectrum (m/e = 774). To determine the source of the copper coproporphyrin a series of tests were performed i n which a VGH-2 f e c a l sample and/or a free porphyrin were e s t e r i f i e d with methanol/H^SO^ or methanol/BF^.  After separation  of the reaction mixture by solvent A or B TLC the r e l a t i v e concentrations were determined spectrophotometrically.  H.  IDENTIFICATION OF THE SUB-URO PORPHYRINS Hematoporphyrin IX and several HPLC and column chromatography i s o -  42 lated sub-uro porphyrin fractions were acetylated by known methods Attempts to obtain mass spectra of the acetylated products were largely unsuccessful.  Hematoporphyrin IX gave an m/e f o r protoporphyrin IX. 43  These same samples were also s y l i l a t e d by known methods  . We were  unable to obtain a mass spectrum of the hematoporphyrin sily.l ether and TLC (benzene/ethyl acetate/methanol  (30/20/2)) analysis of the s y l i l a t e d . reaction  on the sub-uro i s o l a t e s showed a s i g n i f i c a n t decrease i n Rf after reaction.  36  III. A.  RESULTS INVESTIGATION OF HIGH PRESSURE LIQUID, CHROMATOGRAPHIC PARAMETERS FOR THE OPTIMIZATION OF PORPHYRIN ANALYSIS A v a r i e t y of HPLC column/solvent systems were investigated to  determine which combination would give acceptible component resolution and minimum analysis time.  The f e a s i b i l i t y of an i s o c r a t i c (one solvent com-  bination) solvent system was  extensively studied because application of  i s o c r a t i c e l u t i o n to routine analysis i s preferable to solvent programming techniques.  1.  URO  TO PROTO PORPHYRINS  a.  PORPHYRIN FREE ACIDS  The evaluation of two packings for the chromatography of porphyrin free acids did not y i e l d a s a t i s f a c t o r y system. The elution of protoporphyrin  IX from polyamide was very slow with  a l l of the solvent combinations tested which resulted i n unacceptably high values for k'.  The C-18  reverse phase packing gave broad peaks  and/or t a i l i n g (Figure 6) with methanol/water, methanol/2% ammonium hydroxide, acetonitrile/water and a c e t o n i t r i l e / 2 % ammonium hydroxide.  37  -tT  2  TIME (min) FIGURE 6. _  PROTOPORPHYRIN IX ON A C-18 REVERSE PHASE PACKING Solvent:  2% NH.OH/methanol 4  b.  PORPHYRIN ESTERS  i.  ADSORPTION CHROMATOGRAPHY Five adsorption packings were tested with combinations of  methanol, propanol, ethyl acetate, ethyl propanoate, propyl acetate, propyl propanoate, l i g h t petroleum (30-60°) and/or triethylamine i n methylene d i c h l o r i d e , benzene or toluene. Basic alumina had poor s e l e c t i v i t y and very broad peaks 7).  (Figure  38  1  1  4 FIGURE 7.  2  1  TIME (min)  1  4  1  2  PROTOPORPHYRIN IX DIMETHYL ESTER (2) and COPROPORPHYRIN I I I TETRAMETHYL ESTER (4) ON A BASIC ALUMINA PACKING. Solvent:  Methylene  dichloride/n-propanol  N e u t r a l a l u m i n a had good s e l e c t i v i t y but t a i l e d t o o much t o g i v e acceptable  resolution  (Figure 8 ) .  TIME (min) FIGURE 8.  UROPORPHYRIN I I I (8) TO COPROPORPHYRIN I I I (4) METHYL ESTERS ON A NEUTRAL ALUMINA PACKING. Solvent:  Methylene  dichloride(TEA)/methanol  39  P o r a s i l C had good s e l e c t i v i t y but the range of packing p a r t i c l e s i z e resulted  i n extremely broad peaks (Figure 9).  16  12  8  4  .  |  TIME (min) FIGURE 9.  UROPORPHYRIN I I I OCTAMETHYL ESTER ON A PORASIL C PACKING. Solvent:  Methylene dichloride(TEA)/n-propanol  C o r a s i l I I and P o r a s i l T systems gave good o v e r a l l  resolution.  However, even though i t had been determined on neutral alumina that s u b s t i t u t i o n of ethyl propanoate f o r ethyl acetate i n a methylene dichloride/methanol system (Table V H I - a ) or the use of ethyl or propyl esters  (Table VHI-b) s i g n i f i c a n t l y reduced k' f o r the higher carboxy-  lated porphyrins, the e f f e c t was not iarge enough to allow i s o c r a t i c e l u t i o n of a complex sample on e i t h e r C o r a s i l I I or P o r a s i l T. fore, a methylene d i c h l o r i d e / l i g h t petroleum  There-  (30-60)/n-propanol/triethylamine  40  TABLE VHI-a A COMPARISON OF THE EFFECT OF ETHYL ACETATE AND ETHYL PROPANOATE ON PORPHYRIN RETENTION (k')  Solvent  Sample  Ethyl propanoate k'(%)  Ethyl acetate k'(%) Coproporphyrin  2.0 (100)  2.0 (100)  Pentacarboxylic acid  3.1 (100)  2.9 (94)  Hexacarboxylic  5.2 (100)  4.6 (89)  a.  acid  1% of ester and .2% methanol i n methylene d i c h l o r i d e  TABLE V l l l - b A COMPARISON OF THE RELATIVE VARIATION IN RETENTION (k') WITH PROPYL, ETHYL AND METHYL PORPHYRIN ESTERIFICATION Porphyrin Methyl k'(%)  3  Ester Ethyl k' (%) 1.7(94)  Protoporphyrin  1.6(89)  1.8 (100) 4.6(84)  Coproporphyrin 5.5 (100) a.  Solvent:  Propyl k'(%)  .15% methanol i n methylene dichloride  3.5 (63)  41  system was chosen because i t was found to be applicable to the simple step gradient i l l u s t r a t e d by the 254 nm baseline i n f i g u r e 10. e l e c t r o n i c programmer were a v a i l a b l e methylene  If an  dichloride/n-propanol  (.3-1.0% n-propanol) would probably give a good chromatogram.  However,  f o r routine c l i n i c a l analysis the o p e r a t i o n a l l y simpler system seemed best.  35  30  FIGURE 10.  I  25  1  20  — r —  15  time (min)  10  "T~  5  0  ILLUSTRATION OF THE SOLVENT GRADIENT (254 nm). The spike at -2 minutes marks the change to lower p o l a r i t y solvent. The sample i s injected at 0 minutes with the solvent front at 1.5 minutes. The higher p o l a r i t y solvent begins to re-elute at 11 minutes. The peak at 20 minutes i s npropanol/TEA which i s swept from the column by the lower p o l a r i t y solvent.  42  Chromatograms produced by test samples on C o r a s i l I I and P o r a s i l T. (Figures 11a and b) demonstrate the r e s o l u t i o n obtained by HPLC i n comparison to TLC or extraction techniques  (see f i g u r e 3). An added ad-  vantage of the HPLC procedure i s one step e l u t i o n and quantitation which 44 can be further s i m p l i f i e d by the use of an e l e c t r o n i c processor  . Note  that C o r a s i l I I gives maximal r e s o l u t i o n i n the 2-4 carboxyl region and P o r a s i l T gives maximal r e s o l u t i o n i n the 4-8 carboxyl region.  Corasil II  i s w e l l suited to the analysis of f e c a l samples where the 2-4 carboxyl porphyrins dominate while P o r a s i l T gives better r e s o l u t i o n of the 4-8 carboxyl porphyrins which occur predominantly i n urine.  For the routine  needs of a c l i n i c a l laboratory e i t h e r column i s equally s u i t a b l e .  1  20  1  15  1  10  1  5  TlME(min) FIGURE 11a.  REFERENCE CHROMATOGRAM OF URO TO PROTO PORPHYRIN (8-2) METHYL ESTERS ON CORASIL I I .  43  20~  15  TIME(min)  FIGURE l i b .  i5  5  REFERENCE CHROMATOGRAM OF URO TO PROTO PORPHYRIN (8-2) METHYL ESTERS ON PORASIL T. The retention time f o r 3 was determined separately.  ii.  GEL PERMEATION CHROMATOGRAPHY Chromatography with a Sephadex LH-20 g e l gave f a i r r e s o l u t i o n of a  simple porphyrin mixture (Figure 12). To increase the r e s o l u t i o n f o r analysis of a complex mixture would require increased analysis time and a small p a r t i c l e packing.  Unfortunately,  the a v a i l a b l e small p a r t i c l e 45  packings are not compatible with methanol  . The use of a polar solvent i s  necessary as adsorption of the porphyrin ester does play a r o l e i n por46 phyrin g e l chromatography  . Therefore  t h i s system cannot be further developed.  44  19  17  15  13  TIME(min)  FIGURE 12.  SEPARATION OF URO (8), COPRO (4) AND PROTOPORPHYRIN (2) METHYL ESTERS ON SEPHADEX LH-20. Solvent:  Methylene dichloride/methanol  2.  SUB-URO PORPHYRINS  a.  ADSORPTION CHROMATOGRAPHY Methylene dichloride/n-propanol and methylene dichloride/methanol  were used f o r the chromatographic analysis of the sub-uro porphyrins on C o r a s i l I I .  Both systems gave poor r e s o l u t i o n as a r e s u l t of broad peaks.  See section III.C.2. c (P. 57) f o r representative chromatograms.  45  b.  GEL PERMEATION CHROMATOGRAPHY  Sephadex LH-20 with methylene dichloride/methanol did not s i g n i f i c a n t l y separate the sub-uro porphyrins as t h i s heterogeneous group of compounds was  too complex for s i g n i f i c a n t resolution. See Section III.C.2.a  (P. 50)  for representative chromatograms.  B.  SAMPLE PREPARATION PROCEDURES  1.  URINE SAMPLES  The t a l c extraction procedure for urinary porphyrins as outlined i n the introduction requires r e p e t i t i o n reproducible r e s u l t s .  of many i n e f f i c i e n t steps to obtain  Therefore, we modified a procedure based on f l o r i s i l  chromatography recently developed by Schwartz for the separation of por38  phyrins from other urinary components  . This method does not require  tedious repetition or long reaction times to ensure complete extraction 4 and e s t e r i f i c a t i o n . After adsorption of the porphyrins from a c i d i f i e d urine and washing to remove non-porphyric  contaminants the Schwartz method used buffers  based on ammonium ion for porphyrin elution. results  However, we achieved better  i n the diazomethane e s t e r i f i c a t i o n step when an ethanolic T r i s  buffer was used.  The e s t e r i f i c a t i o n with diazomethane immediately  con-  verted a l l of the carboxyl groups to their methyl esters which avoided the time and y i e l d problems inherent with overnight methanol/H^SO^ 4 e s t e r i f i c a t i o n . The f u l l y e s t e r i f i e d porphyrins were extracted from 'the  46  aqueous phase with ether and after solvent removal the sample was ready for HPLC analysis.  Examination of the r e s i d u a l aqueous phase a f t e r  porphyrin extraction, by excitation at 366 nm, showed no residual fluorescence.  2.  FECAL SAMPLES  The t o t a l analysis of porphyric f e c a l samples requires repetitious extraction procedures for removal of "contaminating" proto and copro porphyrin followed by overnight urea/Triton extraction for the sub-uro 11 porphyrins  and analysis of a second sample with methanol/BF^ extraction/  4 e s t e r i f i c a t i o n and TLC for the uro to proto porphyrins .  This duplication  of e f f o r t has been completely eliminated by combining the more e f f i c i e n t methanol/BF^ procedure with column chromatography  to obtain from one sample  fractions suitable for the analysis of both uro to proto and sub-uro porphyrins. The column chromatography  procedure removes non-porphyric impurities  and cleanly separates the uro to proto porphyrins f r a c t i o n from the suburo porphyrin f r a c t i o n .  I n i t i a l l y the column was eluted with methylene  d i c h l o r i d e / e t h y l acetate/methanol but entrainment of uro to proto porphyrins i n the sub-uro porphyrin f r a c t i o n necessitated the use of a benzene/ethyl acetate/methanol system. The benzene/ethyl acetate/methanol column fractionation of the f e c a l porphyrins results i n the loss of a s i g n i f i c a n t portion of the sub-uro material through i r r e v e r s i b l e adsorption to the s i l i c a g e l . The data  47  obtained  from the l a r g e VGH-lb sample i n d i c a t e s t h i s l o s s (based on the  t o t a l Soret absorption of the o r i g i n a l e x t r a c t ) to be $5%.  However,  subsequent a n a l y s i s of the methanol/BF^ e x t r a c t by g e l chromatography ( s e c t i o n III.C.2.a.,P. 50) and e x t r a c t i v e a n a l y s i s of selected f e c a l samples by the Rimington m e t h o d  11  ( s e c t i o n I I I . C . 2 . c . i . , P.57) has shown that the  eluted f r a c t i o n i s r e p r e s e n t a t i v e w i t h respect t o the o r i g i n a l quantity of sub-uro porphyrins present. Fraction  % of t o t a l Soret  Uro to proto porphyrins  34  Eluted sub-uro porphyrins  18  Remaining sub-uro porphyrins (by d i f f e r e n c e )  48  C.  SAMPLE ANALYSIS  1.  URINE SAMPLES The r e s u l t s o f the f l o r i s i l / P o r a s i l T a n a l y s i s of 5 u r i n e samples  are presented i n f i g u r e s 13a and b and t a b l e IX.  TIME(min) FIGURE 13a.  CHROMATOGRAM OF URINE SAMPLE 5 (TABLE I X ) .  48  TIME(min) FIGURE 13b.  CHROMATOGRAM OF URINE SAMPLE 4 (TABLE IX).  TABLE IX RESULTS OF THE HPLC ANALYSIS OF PORPHYRIC URINE Porphyrin a n a l y s i s :  Microgram/litre of urine ( r e l a t i v e percent)  Sample  Number of carboxyl groups 3  4  5  1  4  25 (1)  5  8  190 (45)  180 (43)  60 (10)  230 (39)  270 (47)  5,600 (48) 1,290 ( I D  60 (1)  440 (4)  2,950 (34)  2,150 (40)  50 (1)  280 (5)  2,220 (41)  24 (4) 190 (2)  7  50 (12)  2 3  6  660 (12) 60 (4)  100 (6)  520 (34)  870 (56)  50 or 100 y l of each sample chromatographed. Samples 1, 2 and 5 were from symptomatic porphyria human urine and samples 3 and 4 were from congenital erythropoietic porphyria bovine urine.  49  Comparison of these r e s u l t s with the general c h a r a c t e r i s t i c s outl i n e d i n table V and the l i t e r a t u r e values presented i n figure 14 i n d i c a t e that the data from HPLC analysis are consistent with the values obtained by standard extraction and TLC techniques.  Thus HPLC analysis can pro-  vide diagnostic c l i n i c a l information.  SYMPTOMATIC PORPHYRIA  CONGENITAL ERYTHROPOIETIC PORPHYRIA HPLC (2 samples)  HPLC (3 samples) 5-3%  3-2%  6-9%  4-44%  7-39%  5-11%  8-49%  6-1% 7-4%  5 6 7 8  CHU  47  48  (7 samples)  CARDINAL  4-2%  4-32%  5-3%  5-8%  6-3%  6-1%  7-23%  7-1%  8-67%  FIGURE 14.  3 4 5 6 7 8  8-38%  4 5 6 7 8  8-50%  (2 samples)  h  4 5 6 7 8  COMPARISON OF THE HPLC AND LITERATURE ANALYSIS OF SYMPTOMATIC AND CONGENITAL ERYTHROPOIETIC PORPHYRIA SAMPLES. The bar graphs represent r e l a t i v e per cent of the t o t a l porphyrin sample of the uro to t r i c a r b o x y l i c acid porphyrins (8-3).  50  Also, the HPLC method Is preferable:: to the standard  techniques  because i t i s simpler to use once the system i s set up, requires much less d i r e c t technician time, i s reproducible by d i f f e r e n t analysts and gives a much better separation and quantitation of the porphyrin components.  2.  FECAL SAMPLES  a.  GEL CHROMATOGRAPHIC ANALYSIS  The use of gel chromatography for the analysis of the methylene dichloride extract a f t e r methanol/BF^ e x t r a c t i o n / e s t e r i f i c a t i o n did not give quantitative (baseline) separation of the uro to proto or the sub-uro porphyrins.  porphyrins  However, the chromatograms obtained  (Figure  15) can be used to determine the r e l a t i v e quantities of the proto, copro and most importantly the sub-uro porphyrins at a glance.  51  Proto  Sub-uro Copro  VGH-1a  VGH-2a  SA-b  +  20  15  10  T I M E (min)  FIGURE 15.  HPLC GEL CHR0MAT0GRAMS OF SELECTED FECAL METHYLENE DICHLORIDE EXTRACTS. 50 u l of each sample i n j e c t e d .  400 nm absorbance to scale.  52  The greatly increased sub-uro region of the VGH-la sample chromatogram with respect to the VGH-2a and SA-b samples substantiates the use of the column chromatography sub-uro f r a c t i o n to q u a l i t a t i v e l y determine increased excretion of the sub-uro group.  The low, f l a t zone  of increase seen throughout the whole sub-uro region (ca. 10-15 min) for the VGH-2a and SA-b samples supports the theory suggested by Rimington 49 and others  that the increase i n sub-uro excretion associated with  Variegate porphyria does not represent de novo synthesis but instead i s merely an increased accumulation i n other samples.  and excretion of porphyrins  present  I f the small quantity of sub-uro material observed i n  the VGH-2a and SA-b samples had been of s i g n i f i c a n t l y d i f f e r e n t o r i g i n (eg:  Hydroxyethylisocoporporphyrin)  the chromatogram would have shown  a peak instead of the zone of increased 400 nm absorption.  b.  URO TO PROTO PORPHYRINS The results of the Corasil II HPLC analysis of 5 f e c a l samples  from 3 porphyric patients are presented  i n figure 16 and table X.  —i  20  :  1  r  1  15 .  .  .  N  time (mm)  10  5  FIGURE 16. URO TO PROTOPORPHYRIN (8-2) CHROMATOGRAMS OF FECAL SAMPLES SA-a AND VGH-la. (Table X ) .  54  TABLE X RESULTS OF THE HPLC ANALYSIS OF PORPHYRIC FECES URO Porphyrin analysis:  microgram/gram dry weight feces (relative per cent).  Sample  Number of Carboxyl Groups 2  VGH-la  TO PROTO PORPHYRINS  820  (30)  3  4.  290 (10)  5  7  6  1340  (49)  61 (2)  140  (5)  P  (D  8  42 (2)  52 (2)  VGH-2a  77 (6)  330 (24)  a  950  (68)  26 (2)  VGH-2b  180 (8)  340 (15)  a  1650  (75)  P (0)  25 (1)  SA-a  850 (76)  83 (7)  P (2)  43 (4)  47 (4)  P (1)  SA-b  770 (72)  34 (3)  49 (5)  P (1)  105  (10)  a.  Copper Coproporphyrin  b.  Present (10-20 yg/g)  66 (6) 93 (9)  b  Analysis of the porphyrins i n the t r i c a r b o x y l i c acid porphyrin region of samples VGH-1  and VGH-2 by HPLC/stop-flow v i s i b l e spectroscopy  proved that they were d i f f e r e n t compounds and provided on example of the a n a l y t i c a l c a p a b i l i t y and v e r s a t i l i t y of the HPLC/Cary 17 system. porphyrin spectrum compound of VGH-1  The aetio  was found by mass spectral analysis to  be harderoporphyrin and/or isoharderoporphyrin and related products from microbial a c t i v i t y i n the gut.  The compound with comparable  chromatographic  retention from VGH-2 had a metalloporphyrin spectrum and was shown to be copper coproporphyrin.  Analysis of suitable test samples by TLC and  55  spectroscopy (Table XI) proved that the copper coproporphyrin was not formed during the e s t e r i f i c a t i o n / e x t r a c t i o n procedure either  from  33 extraneously introduced copper  or from copper present i n the feces but  not combined with coproporphyrin. TABLE XI  COPPER COPROPORPHYRIN TEST SAMPLES  Sample  Esterification  TLC  Result  a  b  Feces  MeOH/BF  A  Cu-Copro (+)  Feces  Me0H/H S0, z 4  A  Cu-Copro (+)  Copro  Me0H/BF  A  Cu-Copro (-)  Proto  Me0H/BF  B  Cu-Proto (-)  Feces + Copro  MeOH/BF^  A  Cu-Copro (+)  Feces + Proto  MeOH/BF.  A B  Cu-Copro (+) Cu-Proto (-)  a.  3  o  3  3  Copper protoporphyrin and copper coproporphyrin had Rf. .35 and .17  respectively i n solvent B. b.  (+) present:  Soret; (-) absent:  copper coproporphyrin Soret ca. 25% of coproporphyrin copper coproporphyrin Soret «  1% of coproporphyrin  Soret. c.  Greatly reduced r e l a t i v e to the t o t a l coproporphyrin.  C  56  2 48 A review of the l i t e r a t u r e ' revealed that an unidentified copper porphyrin and/or a pink,non^fluorescent band has been observed  i n the  appropriate position i n the TLC analysis of urine and f e c a l samples 2 (especially those samples from Hereditary coproporphyrics ). The b i o l o 13 27 g i c a l r o l e of t h i s compound i s unknown ' although i t i s probably excreted 27 to the feces v i a the b i l e The VGH samples were obtained l o c a l l y so a comparison of the 27 clinical  and HPLC data was possible.  Patient VGH-2 was diagnosed  as a  Hereditary coproporphyric on the basis of her c l i n i c a l presentation and laboratory analysis.  The HPLC r e s u l t s agree very well (sample VGH-2a) when Clinical analysis  .:. Protoporphyrin (yg/g) Coproporphyrin  (yg/g)  HPLC analysis  74  77  1270  1280  the values f o r copper coproporphyrin and coproporphyrin are combined. other VGH patient (VGH-1) was diagnosed extensive porphyrin analysis.  The  as a Variegate porphyric without  The HPLC values (when combined with sub-  uro analysis; see table V) substantiate this diagnosis. Unfortunately, the c l i n i c a l analysis of patient SA was unavailable. Our results indicate that t h i s sample represents a case of Erythrohepatic protoporphyria.  57  C.  SUB-URO PORPHYRINS  i.  SCREENING PROCEDURES  The sub-uro f r a c t i o n obtained from the column chromatographic  pre-  paration of porphyric f e c a l samples was used to q u a l i t a t i v e l y determine the increase i n the sub-uro porphyrins by measuring the corrected Soret absorption of the sample. Soret absorptions  The r e l a t i v e difference between corrected  (Table XII), the values obtained by the Rimington method  (Table XII) and the gel chromatographic III.C.2.a, P.50)  data presented previously (Section  i l l u s t r a t e that the column procedure, which i n one step  y i e l d s the uro to proto porphyrins and the sub-uro f r a c t i o n , can be u t i l i z e d for screening sub-uro porphyrin l e v e l s .  TABLE XII SPECTROSCOPIC ANALYSIS OF THE SUB-URO PORPHYRIN FRACTION BY THE COLUMN AND RIMINGTON PROCEDURES Sample  Corrected Soret  Rimington  absorption  ug/g  VGH-.la  .91  2300  VGH-lb  .79  VGH-2a  .07  VGH-2b  .10  SA-a  .11  SA-b  .09  The VGH-1  and 2 r e s u l t s correlate  13 Rimington ... and Elder et a l  140  94 well with table V and the work of  49 which indicate a greatly increased l e v e l of sub-  uro porphyrins for Variegate porphyria and a moderately Hereditary coproporphyria.  increased;level for  58  ii.  CHROMATOGRAPHY AND IDENTIFICATION Analysis of the sub-uro porphyrin column f r a c t i o n by HPLC provided  a d d i t i o n a l information on the composition and source of the sub-uro group. Although the chromatograms (Figures 17 and 18) represent only ca. 15%  8  4 TIME(min)  FIGURE 17.  CHROMATOGRAPHIC  ILLUSTRATION OF THE QUALITATIVE SLMILARIES  OF THE FECAL-SUB-URO PORPHYRINS; 1.50% METHANOL IN METHYLENE DICHLORIDE ANALYSIS.  59  of the t o t a l sub-uro porphyrins o r i g i n a l l y present i n the methylene d i c h l o r i d e extract they i l l u s t r a t e that the f r a c t i o n obtained from samples with large quantities of the sub-uro porphyrins i s of e s s e n t i a l l y  -\ 8  FIGURE 18.  CHROMATOGRAPHIC  :  TIME(min)  h 4  ILLUSTRATION OF THE QUALITATIVE SIMILARITIES  OF THE FECAL SUB-URO PORPHYRINS; 1.00% METHANOL IN METHYLENE DICHLORIDE ANALYSIS.  60  the same composition  (porphyrins A, B, C, D) as similar fractions from  samples with much smaller quantities of the sub-uro porphyrins. A, B  The sub-uro  fractions obtained from the large- sub-uro porphyrin extraction  were used as standards to prove that porphyrins A and B on the chromatograms :  were d i f f e r e n t compounds.  Porphyrin A i s present i n the 1.50% methanol i n  methylene dichloride chromatogram as the solvent front shoulder on porphyrinoB. A d d i t i o n a l l y , figure 19 demonstrates the use of a v a r i a b l e wavelength and scanning HPLC detector to determine the porphyrin composition of a complex sample.  Porphyrin A i s the only porphyrin of the 5 peaks  recorded i n the 254 nm spectrum. Various sub-uro porphyrin samples were acetylated and/or s y l i l a t e d for further chromatographic  and structure analysis.  samples exhibited an increase i n chromatographic  The acetylated  mobility (TLC and HPLC)  for some compounds indicating the presence of reactive hydroxyls but the samples were not v o l a t i l e enough for massr: spectral analysis.  The  s j l y l a t i o n reaction formed products which decreased substantially i n TLC Rf.  Therefore mass spectra were not attempted.  61  STOP-FLOW FIGURE 19.  S C A N (nm)  ILLUSTRATION OF THE USE OF VARIABLE WAVELENGTH DETECTION AND STOP-FLOW VISIBLE SCANS FOR SUB-URO PORPHYRIN ANALYSIS. V i s scan:  460-590 nm region scaled 2.5 x Soret region.  62  IV.  CONCLUSION  The evaluation of a variety of e s t e r i f i c a t i o n / e x t r a c t i o n procedures and HPLC packing and solvent;systems f o r the analysis of porphyrins from porphyric urine and f e c a l samples has l e d to the development of procedures using s i l i c a based supports which provide an alternative to the standard extractive and TLC techniques currently used f o r c l i n i c a l and biochemical analysis.  The standard techniques are tedious  and i n e f f i c i e n t which r e s u l t s i n unnecessary  27 expense' and variable  48 analytical results  . Also, the analysis of trace or unusual metabolic 9 products usually requires preparation of a separate sample or may even 11 27  be impossible on a routine bases  '  . HPLC, on the other hand, has  been shown to simply and e f f i c i e n t l y provide a n a l y t i c a l l r e s u l t s f o r both routine and complex analyses.  We diagnosed patient VGH-1 as a  Variegate porphyric by uro to proto (and sub-uro) porphyrin analysis of a single f e c a l sample.  In contrast, her. . c l i n i c a l laboratory b i l l was i n  27 excess of $6,000  . The a b i l i t y of the HPLC/Cary 17 system to i d e n t i f y  unusual components was demonstrated by the characterization of the TLC similar hardero-type and copper coproporphyria. The standard techniques currently i n use also have the d i s t i n c t disadvantage of requiring a separate procedure f o r each type of sample. Besides the demonstrated f e c a l preparation the use of methanol/BF^ should provide extracts suitable for HPLC analysis from such diverse sources.'ias blood, l i v e r and cultured tissue.  Also, the experience gained from 17 35 the HPLC optimization analysis i n combination with recent reports ' on  63  superior porphyrin r e s o l u t i o n with microparticulate packings  (5-10um)  should allow the development of a single, convenient i s o c r a t i c HPLC analytical  procedure.  Perhaps the most s i g n i f i c a n t proofs of the advantage of the procedures reported here are the current plan of the c l i n i c a l laboratory which provided the VGH samples to purchase an HPLC system for porphyrin analysis and the formation of a j o i n t c l i n i c a l , b i o c h e m i c a l and chemical team to u t i l i z e HPLC analysis of porphyric tissue cultures f o r determination of the enzymic defects which cause porphyric disease.  This  study requires the analysis of porphyrinogens as well as porphyrins; a procedure which was extremely d i f f i c u l t or impossible before the advent of anaerobic, light-proof HPLC. Analysis of the f e c a l sub-uro porphyrins has been shown to be of use i n the diagnosis of some p o r p h y r i a s . 11  Unfortunately, the procedures  presently a v a i l a b l e for their determination are too involved to be of  27 much use i n a c l i n i c a l laboratory  and have been unable to give more  than q u a l i t a t i v e information on the structure of the compounds present (eg:  they are mostly protein porphyrin complexes). We have developed  open column and HPLC gel column procedures f o r  the c l i n i c a l analysis of this group.  Although neither procedure  currently provides quantitative information the e a s i l y obtained data has been shown to be suitable for the determination of r e l a t i v e sub-uro porphyrin increase.  64  The most s i g n i f i c a n t aspect of t h i s work with respect to research on heme metabolism as well as porphyria has been our prelminary  success  i n the separation and i d e n t i f i c a t i o n of the protein porphyrin complex/ sub-uro porphyrins.  Although l i t t l e exact s t r u c t u r a l information was  obtained we found q u a l i t a t i v e s i m i l a r i t i e s i n the content of samples from d i f f e r e n t sources.  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