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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 in 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 presenting th i s thesis in pa r t i a l fu l f i lment of the requirements for o an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f ree ly ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th i s thes is for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i ca t ion of th is thes is fo r f i nanc ia l gain sha l l not be allowed without my wr i t ten permiss ion. Department of TnJTS r The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date ' 2Q jL4> . / ? f-C 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. The c l i n i c a l evaluation and sub-sequent treatment of porphyria are dependent on the analysis of these compounds in urine, feces and blood because a wide array of overlapping physiological manifestations make direct c l i n i c a l diagnosis d i f f i c u l t . The analytical techniques currently available are inefficient and do not have the capacity to separate or easily quantify complex mixtures of porphyrins. High pressure liquid chromatography (HPLC), on the other hand, can readily achieve complex separations and since the sample being analyzed remains in solution at a l l times i t s detection and quantitation are considerably simplified. Bearing these considerations in mind, we have developed new porphyrin isolation 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 efficient and should be easily extended to other porphyric samples. The ve r s a t i l i t y of the HPLC system was demonstrated by the characterization of copper coproporphyrin from a porphyric fecal sample.. We have also developed procedures for the qualitative analysis of the fecal sub-uro porphyrins and made prelminary progress toward the separation and characterization of this heterogeneous group of compounds., i i CONTENTS SECTION PAGE Abstract i Contents ' . . • . • 1 1 L i s t of Tables v List of Figures v i Acknowledgements . v i i i Dedication -% • l x I. INTRODUCTION 1 A. Cl i n i c a l and biochemical aspects of porphyria 1 1. Porphyrin metabolism 1 a. Porphyrin structure 2 b. Porphyrin biosynthesis 5 c. Porphyrin excretion 7. 2. Classification of the porphyrias: Cl i n i c a l and biochemical symptoms 9 B. Current " analytical methods for the determination of porphyrins and porphyrin precursors in c l i n i c a l samples 14 1. Urine samples 14 a. Porphyrin precursors 14 b. Uro to proto porphyrins 14 2. Fecal samples 15 a. Uro to proto porphyrins 15 b. Sub-uro porphyrins 18 3. Blood samples 19 i i i CONTENTS, continued. SECTION PAGE C. . HPLC background 21 1. Resolution theory 21 2. Separation modes 22 3. Porphyrin HPLC 22 II. EXPERIMENTAL 24 A. HPLC instrumentation 24 1. The high pressure liquid chromatograph 24 2. Special apparatus: A variable wavelength and scanning detector 25 B. Solvent and column preparation 26 1. Solvents 26 2. Columns 26 C. Porphyrin identification 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 D. Pre-analysis preparation of porphyric samples 28 1. Urine samples 29 2. Fecal samples 30 E. Spectroscopic analysis of the sub-uro fraction 32 F. HPLC of c l i n i c a l samples 33 1. Urine samples 33 CONTENTS, continued. i v SECTION PAGE 2. Fecal samples 33 a. Uro to proto porphyrins 33 b. Sub-uro porphyrins 33 G. Identification of the "tricarboxylic acid porphyrins" .. 34 1. Analogues of harderoporphyrin 34 2. Copper coproporphyrin 34 H. Identification of the sub-uro porphyrins 35 III. RESULTS 36 A. Investigation of high pressure liquid chromatographic parameters for the optimization of porphyrin analysis 36 1. Uro to proto porphyrins 36 a. Porphyrin free acids 36 b. Porphyrin esters 37 i . Adsorption chromatography 37 i i . Gel (permeation) chromatography 43 2. Sub-uro porphyrins 44 a. Adsorption chromatography 44 b. Gel (permeation) chromatography 45 B. Sample preparation procedures 45 1. Urine samples 45 2. Fecal samples 46 C. Sample analysis 47 1. Urine samples 47 2. Fecal samples 50 a. Gel chromatographic analysis 50 b. Uro to proto porphyrins 52 c. Sub-uro porphyrins 57 i . Screening procedures 57 i i . Chromatography and identification 58 IV. CONCLUSION 62 BIBLIOGRAPHY 65 V LIST OF TABLES TABLE PAGE I Porphyrins referred to in this study 4 II Porphyrins found in porphyric samples 8 III Classification of the porphyrias 9 IV Main c l i n i c a l features of the porphyrias 10 V Cli n i c a l features of the acute attack: A comparison of percentage incidence 11 VI Values for urinary, fecal and erythrocyte porphyrins and porphyrin precursors in porphyric and normal patients 13 VII Description of the HPLC column packings used in this study 27 VIII a. A comparison of the effect of ethyl acetate and ethyl propanoate on porphyrin retention (k') 40 VIII b. A comparison of the relative variation in retention (k') with propyl, ethyl and methyl porphyrin esterification 40 IX Results of the HPLC analysis of porphyric urine 47 X Results of the HPLC analysis of porphyric feces uro to proto porphyrins 54 XI Copper coproporphyrin test samples 55 XII Spectroscopic analysis of the sub-uro porphyrin fraction by the column and Rimington procedures 57 v i LIST OF FIGURES FIGURES PAGE 1. Uroporphyrinogen I II metabolism 6 2. Regions of the heme biosynthetic pathway affected i n the porphyrias 12 3a. TLC analysis of acid/ether extracted proto and copro f r a c t i o n s from porphyric feces 16 3b. TLC/chromatoscan chromatograph of a porphyric sample 17 4. Schematic of an HPLC system 24 5. Diagram of the HPLC/Cary 17 flow c e l l 25 6. Protoporphyrin IX on a C-18 reverse phase packing 37 7. Protoporphyrin IX dimethyl ester (2) and coproporphyrin I I I tetramethyl ester (4) on a basic alumina packing 38 8. Uroporphyrin I I I (8) to coproporphyrin I I I (4) methyl esters on a neutral alumina packing 38 9. Uroporphyrin I II octamethyl ester 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 of the solvent gradient (254 nm) 41 11a. Reference chromatogram of uro to proto porphyrin (8-2) methyl esters on C o r a s i l II 42 l i b . Reference chromatogram of uro to proto porphyrin (8-2) methyl esters on P o r a s i l T 43 12. Separation of uro (8), copro (4) and protoporphyrin (2) methyl esters on Sephadex LH-20 44 13a. Chromatogram of urine sample 5 (Table IX) 47 13b. Chromatogram of urine sample 4 (Table IX) 48 14. Comparison of the HPLC and l i t e r a t u r e analysis of Symptomatic and Congenital erythropoietic porphyria samples 49 15. HPLC gel chromatograms of selected f e c a l methylene' d i c h l o r i d e extracts 51 v i i FIGURES, continued. FIGURES PAGE 16. Uro to protoporphyrin (8-2) chromatograms of fecal samples SA-a and VGH-la (Table X) 53 17. Chromatographic i l l u s t r a t i o n of the qualitative similarities of the fecal sub-uro porphyrins; 1.50% methanol in methylene dichloride analysis 58 18. Chromatographic i l l u s t r a t i o n of the qualitative similarities of the fecal sub-uro porphyrins; 1.00% methanol in methylene dichloride analysis 59 19. Illustration of the use of variable wavelength detection and stop-flow visible scans for sub-uro porphyrin analysis 61 v i i i THIS THESIS WOULD BE INCOMPLETE WITHOUT AN EXPRESSION OF THANKS TO: Dr. David Dolphin 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 the forest from the trees. Dr. Rozanne Poulsen for a review of t h i s t h e s i s . Dr. Melvin Bernstein f o r providing c l i n i c a l information and samples. Dr. Sam Schwartz f o r providing c l i n i c a l samples and h i s f l o r i s i l procedure. i x TO John and l i n d a and most of a l l 1 I. INTRODUCTION The porphyrias are a group of acquired or inherited metabolic diseases characterized by the abnormal production and excretion of por-phyrins 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 in urine, feces and blood because a wide array of overlapping physiological manifestations make direct c l i n i c a l 1 2 3 diagnosis d i f f i c u l t ' ' . The analytical techniques currently available are inefficient and do not have the capacity to separate or easily quantify complex mixtures 4 5 of porphyrins ' . High pressure liquid 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 quan-titation are considerably simplified. Bearing these considerations in mind, we have coupled new isolation techniques with HPLC and developed rapid and efficient methods for the analysis of porphyrins in urine and feces. A. CLINICAL AND BIOCHEMICAL ASPECTS OF PORPHYRIA 1. PORPHYRIN METABOLISM Porphyrins play important roles in many biological processes from oxygen and electron transport to drug detoxification^. Because of the variation in porphyrin structure and function this introduction considers only those compounds important to heme biosynthesis and the study of porphyrin excretion in porphyria. 2 The patterns of porphyrin excretion i n porphyric patients directly reflects 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 identifying the underlying enzymic defects which are expressed as porphyric disease. a. PORPHYRIN STRUCTURE The porphyrin macrocycle occurs in 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 substitution patterns encountered in this study. The three most common compounds from this group are: P A UROPORPHYRIN III (URO III) P M P P COPROPORPHYRIN III (COPRO III) V M P P PROTOPORPHYRIN IX (PROTO) TABLE 1 PORPHYRINS REFERRED TO IN THIS STUDY 1,8,9,10,11,12 PORPHYRIN UROPORPHYRIN I I I UROPORPHYRIN I 11EPTACARBOXYIIC ACID PORPHYRIN I I I HEPTACARBOXYLIC ACID PORPHYRIN I c HEXACARBOXYLIC ACID PORPHYRIN I I I HEXACARBOXYLIC ACID PORPHYRIN I c PENTACARBOXYLIC ACID PORPHYRIN I I I PENTACAREOXYLIC ACID PORPHYRIN I c 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 ••' CLASS POSITIONS 1 2 3 A 5 6 7 8 UP A P A P A P P A UP A P A P A P A P UP • A P A P A P P M UP A . P A P A P M P UP M P A P A P P M UP M P A P A P M P UP M P M P A P P M UP M P M P A P M P UP M V M P A . P P M UP M E M P A P P M UP M H M P A P P K UP M P M P M P P M UP M P M P M P M P UP M M P M P P M UP M E M P M P P M UP M P M V M P P H UP M P M E M P P M UP M V M V M P P M su M HY M P A P P M su M HY M HY M P P M su M R M R M P P M d. UP: Uroporphyrin to protoporphyrin p o s i t i o n on methyl ester s i l i c a g e l TLC; SU: Sub-uroporphyrin p o s i t i o n on methyl ester s i l i c a g e l TLC. The symbols are: A - -CH 2C0 2H, P = -CH 2CH 2C0 2H, M - -CH 3, V - -CH=CH2, E » -CH 2CH 3, HY •= -CHOH.CH3> R = -CHS (Polypeptide) .CH3, H - -H. Structure not proved: Assumed c o r r e c t by analogy to the I I I system and uro I, copro I. A diverse group of porphyrins which may al s o include monohydroxy-e t h y l , monopolypeptide'-ethyl porphyrins (R(2) or R(4) = HY). 5 b. PORPHYRIN BIOSYNTHESIS 5-aminolaevulinic acid (ALA), the f i r s t compound b i o l o g i c a l l y committed 0 II H 2NCH 2CCH 2CH 2C0 2H 5-Aminolaevulinic acid to porphyrin bios y n t h e s i s , i s derived from acetate v i a the Kreb's c y c l e and «* -oxoglutarate. Two molecules of ALA are condensed by ALA dehydratase to form the monopyrrole porphobilinogen (PBG)'''. Uroporphyrinogen P A kCH 2 NH 2 r M "O N' H Porphobilinogen synthetase causes four molecules of PBG to condense to give the abnormal uroporphyrin isomer: Uroporphyrinogen I. When uroporphyrinogen syn-thetase acts i n the presence of uroporphyrinogen co-synthetase the normal uroporphyrin isomer, uroporphyrinogen I I I , i s formed. Successive decarboxylation of the four carboxymethyl groups of uroporphyrin I to g methyls by uroporphyrinogen decarboxylase gives the corresponding hepta-c a r b o x y l i c a c i d , hexacarboxylic a c i d , pentacarboxylic a c i d and copro-1 8 porphyrinogens I. Coproporphyrinogen I i s not fu r t h e r metabolized ' . The metabolism of uroporphyrinogen I I I i s diagrammed i n fi g u r e 1. Note the presence of normal and abnormal pathways. 6 UROPORPHYRINOGEN III HEPTACARBOXYLIC ACID PORPHYRINOGEN III V HEXACARBOXYLIC ACID PORPHYRINOGEN III PENTACARBOXYLIC ACID PORPHYRINOGEN III Lc DEHYDROISOCOPRO-PORPHYRINOGEN \ \ \ \ \ COPROPORPHYRINOGEN III \ \ \ \ \ \ 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 in vitro FIGURE 1. UROPORPHYRINOGEN III METABOLISM 3' 8 , 1 0' 1 1' 1 3 7 The processes which r e s u l t i n the conversion by coproporphyrino-genase and protogen dehydrogenase^ of coproporphyrinogen I I I v i a proto-11 13 porphyrinogen IX to protoporphyrin IX are not w e l l understood ' In p a r t i c u l a r the formation of a heterogenous group of cysteine t h i o -ether l i n k e d p r o t e i n porphyrin intermediates has been observed i n the i n 11 13 v i t r o coproporphyrinogenase r e a c t i o n ' . A p o s s i b l e example of t h i s group i s d i c y s t e i n y l hematoporphyrin IX"'""'". N H 2 C H 3 H 0 2 C C H C H 2 S C H C H C H 3 S C H 2 C H C 0 2 H |NlH2 DICYSTEINYL HEMATOPORPHYRIN IX C. PORPHYRIN EXCRETION The compounds of normal and abnormal porphyrin metabolism are excreted v i a the urine and feces as porphyrin precursors, porphyrinogens, 1 3 porphyrins and porphyrin metal complexes ' . Because the porphyrinogens are unstable they are analyzed i n the t o t a l porphyrin f r a c t i o n a f t e r o x i -dation. Table II l i s t s the porphyrins which have been observed i n the an a l y s i s of urine and f e c a l specimens. The products of heme catabolism are excreted as non-macrocyclic pigments^'•^'^"''^ and consequently do not play a r o l e i n porphyrin analysis. TABLE II PORPHYRINS FOUND IN PORPHYRIC SAMPLES1'2 ' 1 0 ' 1 : L Uroporphyrin I, III Zinc uroporphyrin Heptacarboxylic acid porphyrin I, III Hexacarboxylic acid porphyrin I, III Pentacarboxylic acid porphyrin I, III Dehydroisocoproporphyrin Isocoproporphyrin'3 Desethylisocoproporphyrin Hydroxyethylisocoproporphyrink Coproporphyrin I, III 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 9 2. CLASSIFICATION OF THE PORPHYRIAS: CLINICAL AND BIOCHEMICAL SYMPTOMS At least nine distinct porphyric diseases are recognized (Table III) 1,3 with physical symptoms spanning the range from minimal discomfort to severe mental i l l n e s s , paralysis and death. TABLE III CLASSIFICATION OF THE PORPHYRIAS E r y t h r o p o i e t i c porphyria Congenital er y t h r o p o i e t i c porphyria (CEP), porphyria congenita, Gunther's disease Erythrohepatic porphyria Erythrohepatic protoporphyria (EHPP), formerly e r y t h r o p o i e t i c protoporphyria Erythrohepatic coproporphyria (EHCP), formerly e r y t h r o p o i e t i c coproporphyria, coproporphyrinemia The hepatic porphyrias Hereditary forms Acute i n t e r m i t t e n t porphyria (AIP), Swedish genetic porphyria, p y r r o l i a , pyrroloporphyria Hereditary coproporphyria (HC), i d i o p a t h i c copropor-p h y r i n u r i a Variegate porphyria (VP), South A f r i c a n genetic porphyria, mixed porphyria, porphyria cutanea tarda h e r e d i t a r i a , protocoproporphyria Symptomatic forms: symptomatic porphyria (SP), por-p h y r i a cutanea tarda symptomatica, acquired porphyria, secondary porphyria, Bantu porphyria, c o n s t i t u t i o n a l porphyria, urocoproporphyria SP associated with alcohol abuse (SP-A) plus i r o n overload SP induced by chemicals (SP-C), hexachlorobenzene (HCB), pentachlorophenol, s t e r o i d drugs and other chemical compounds, and possibly b a c t e r i a l and fungal metabolites SP associated with disease (SP-D), hepatic porphyrinoma, and a miscellaneous group of rare diseases. 10 Unfortunately, the heterogeneity of c l i n i c a l responses (Tables IV and V) which result from the variety as well as the cycl i c a l (acute 3 attack:remission) ; nature of some of the porphyrias make correct diagnosis and subsequent treatment d i f f i c u l t . In addition, the secondary 3 coproporphyrinurias are non-porphyria diseases resulting in abnormal coproporphyrin excretion which further complicates interpretation. TABLE IV MAIN CLINICAL FEATURES OF THE PORPHYRIAS Type of Usual Mode of Mechanical Acute Acute Porphyria Age of Inheritance Dermatosis. Photo- Attacks Onset sensitivity (years) Erythropoietic CEP Congenital; Recessive + + 0 Erythrohepatic EHPP Under 10 Dominant + 0 EHCP Under 10 Dominant + 0 Hepatic AIP Under 30 Dominant 0 0 + HC Under 10 Dominant + ' (+) + VP Under 30 Dominant + (+) + SP-A Over 40 0 + (+) 0 SP-C Any age 0 + (+) 0 SP-D Over 60 0 + (+) 0 Key: + = present,(+) = very rarely present, 0 = absent. 11 TABLE V CLINICAL FEATURES OF THE ACUTE ATTACK: A COMPARISON OF PERCENTAGE INCIDENCE3 Sweden U.K. U.S.A. S. Africa [233] a [50] [69] [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 C 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 — 8 Leukocytosis'1 7 24 48 20 Cranial nerves 1 29 51 9 ECG abnormalities 1 44 47 23 a. Bracketed figures refer to the number of patients in each series. b. May include delirium, hysteria, apathy and epileptiform seizures. c. Especially respiratory paralysis. d. Fever e. Abnormally rapid heart action. f. An excess of nitrogenous bodies in the blood as a result of kidney insufficiency. g. The presence of protein in the urine. h. Increase in the number of blood leukocytes. 12 Therefore, additional information from biochemical analysis i s necessary for diagnostic differentiation. Table VI gives a composite of the normal and abnormal porphyrin and porphyrin precursor patterns which aid in the correct diagnosis of porphyria. Figure 2 indicates the regions of the heme biosynthetic pathway believed to contain the malfunctioning metabolic enzyme(s). This outline i s the result of the study of porphyrin 13 excretion patterns . Additional research i s necessary to identify the exact malfunction responsible for each disease. CONGENITAL PORPHYRIA ALA ACUTE INTERMITTENT PORPHYRIA UROPORPHYRINOGEN II I 1 T HEREDITARY COPROPORPHYRIA VARIEGATE PORPHYRIA UROPORPHYRINOGEN I COPROPORPHYRINOGEN I COPROPORPHYRINOGEN II I . T ERYTHROHEPATIC COPROPORPHYRIA PROTOPORPHYRIN IX SYMPTOMATIC PORPHYRIA ERYTHROHEPATIC PROTOPORPHYRIA FIGURE 2. REGIONS OF THE HEME BIOSYNTHETIC PATHWAY AFFECTED IN THE PORPHYRIAS1'17. TABLE VI 13 VALUES FOR URINARY, FECAL AND ERYTHROCYTE PORPHYRINS AND PORPHYRIN PRECURSORS IN PORPHYRIC AND NORMAL PATIENTS 1 , 3 Type of Porphyria Blood Urine Feces Isomer type Normal Congenital Erythro-p o i e t i c Erythrohepatic Protoporphyria Erythrohepatic Coproporphyria Acute Intermittent Porphyria Hereditary Copro-porphyria Symptomatic Por-phyria Proto 4-52 ug/100 ml Copro 0-4 ug/100 ml Increased (mainly uro) Increased (mainly proto) Increased (mainly copro) Normal Normal Variegate Porphyria Normal Normal ALA 2.5 mg/day Proto 0-76 ug/g PBG 10 mg/day Copro 0-20 ug/g C0PR0 0-160 y g / l i t e r Ether-Insoluble III URO 6-30 y g / l i t e r ALA, PBG - normal URO-much increased COPRO-increased Us u a l l y normal Us u a l l y normal ALA.PBG-increased URO-may be present^ ALA,PEG-may be i n -creased COPRO-increased ALA,PEG-increased e COPRO,URO-may be increased with COPRO greater than URO ALA.PBG-normal URO-large increase Porphyrin 0-22 Vg/g c PROTO-some increase COPRO-increased URO-some increase SUB-URO-not deter-mined PROTO-increased COPRO-sometimes increased Usually normal Normal III I I I COPRO-increased SUB-URO-slight increase III PROTO,COPRO-large Mainly I I I Increase with PROTO greater than COPRO SUB-URO-large i n -crease PROTO,COPRO-moder- Complex ate increase with COPRO greater than PROTO ETHER-INSOLUBLE-in-creased a. ug/100 ml of packed c e l l s b. Ug/g of dry 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 heptacarboxylic a c i d , uro and sub-uro porphyrins d. Uro may be present from the non-enzymatic condensation of PBG e. May be normal during remission 14 B. CURRENT ANALYTICAL METHODS FOR THE DETERMINATION OF PORPHYRINS AND PORPHYRIN PRECURSORS)IN CLINICAL SAMPLES The currently employed analytical methods can be divided into qualitative methods which are useful for screening and preliminary diagnosis and quantitative methods which are a necessity for the positive identification of the specific 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 (2% w/v p-dimethylamino-benzaldehyde in 5 N. HC1) 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 after qualitative porphyrin separation by acidification and extraction with ether"*. This procedure yields 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 glacial acetic adid; and approximately l/10th volume of powdered talc was added. The slurry was stirred for lhn,after which the talc and adsorbed porphyrins were harvested by f i l t r a t i o n with suction. This procedure was repeated until an acetic acid:ether:amyl acohol (1:1:1) extract of the f i l t r a t e no longer fluoresced under ultra-violet light. The talc was then washed with 1% (v/v) aqueous acetic acid and water and thor-oughly dried in a vacuum dessicator over s i l i c a gel. 15 The adsorbed porphyrins were eluted and e s t e r i f i e d i n one step by suspending the t a l c i n methanol: H2SO4 (20:1) and allowing the suspension to stand overnight i n the dark. The suspension was then f i l t e r e d and the t a l c washed with methanol: H2SO4 (40:1) u n t i l i t no longer fluoresced. The pooled f i l t r a t e and washing were adjusted to pH 4.0 with saturated sodium acetate and the porphyrin methyl esters were extracted into chloroform. The chloroform s o l u t i o n • -ii of methyl esters was washed four times with d i s t i l l e d water, and taken to dryness under reduced pressure. The extracted porphyrins are separated by t h i n layer chromatography (TLC) and quantitated i n s i t u with a fluorescence scanner or spectrophoto-2 20 m e t r i c a l l y a f t e r e l u t i o n from the p l a t e ' . See f i g u r e 3b for a representative fluorescence scan of a porphyrin sample separated by TLC. The isomeric composition of a sample (I and/or III) i s determined 21 by the hydrolysis and decarboxylation of the i s o l a t e d uro, heptacarboxylic ac i d , hexacarboxylic acid or pentacarboxylic acid porphyrin to copropor-phyrin followed by overnight chromatography using a paper/lutidene system. The decarboxylation i s necessary because the respective isomeric uro to pentacarboxylic acid porphyrins 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 screening of f e c a l samples also uses an acid/ether p a r t i t i o n followed by a further s e l e c t i v e extraction for separation of protoporphyrin and coproporphyrin f r a c t i o n s ^ . Although t h i s procedure gives useful general information the analysis provides l i t t l e or no i n -formation on the more polar porphyrins (uro, heptacarboxylic acid) and the 16 4 application of TLC to the isolated fractions (Figure 3a) after methanol/R^SO^ esterification il l u s t r a t e s the lack of porphyrin resolution Quantitative analysis i s similar to the TLC method used for urine after extraction of the fecal porphyrins as their methyl esters with 4 methanol/H^SO^ of methanol/BF^ . Figure 3b il l u s t r a t e s the analytical resolution normally obtained with this procedure. If the analysis requires quantitative separation and isolation of specific porphyrin components in the sample additional extraction and/or chromatographic steps are usually 9 20 23 24 25 necessary which greatly add to the time and complexity of analysis ' ' ' . Isomer analysis i s the same as for urine samples. © Proto u ! ft C o p r o FIGURE 3a. TLC ANALYSIS OF ACID/ETHER EXTRACTED PROTO AND COPRO FRACTIONS FROM PORPHYRIC FECES4. 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 qualitatively by observation of 20 26 red-fluorescent bands in the sub-uro area of the developed TLC plate ' or semiquantitatively using the extraction technique of Rimington1"'" outlined here. Ether soluble porphyrin (copro, proto) was removed by six 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) Triton X-100) and then centri-fuged. Extraction was thrice repeated and the combined extracts shaken twice with n-butanol. After washing this solvent; 1 volume of ether and 0.5 volume light 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 Triton which interfered with purification 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 solid phase exhaustively with 50% (v/v) aqueous ethanol. This removed a l l the detergent and the adsorbant could then be redis-solved by addition of a slight excess of dilute HC1. Usually, most of the porphyrin-containing material dissolved readily 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 identified as thio-ether linked protein porphyrins of molecular weight 1000 to 6000"*" 26 or as hydroxyethylisocoproporphyrin the lack of resolution of these components by TLC (Figure 3a) and the extra processing steps necessary 19 to remove ^contaminating Triton X-100 from the Rimington samples before further separation has severely hindered the development of routine analytical procedures. 3. BLOOD SAMPLES The analytical 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 cells thoroughly with 25 ml of ethyl acetate/ acetic-acid - adding the solvent slowly with constant stirr i n g . 3. Allow the mixture to stand until the fine reddish-brown precipitate settles 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 precipitate with three successive 15 ml portions of the extraction mixture allowing the pre-cipitate to settle 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 collecting the washings in a second 250 ml separatory funnel. (Allow at least 10 minutes for 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 un t i l no red fluore-scence can be detected in the acid layer under ultraviolet light. A minimum of two extractions i s required. 20 9. Add saturated sodium acetate to the collected HC1 ex-tracts u n t i l they are basic. 10. Add 2 ml of glacial acetic acid to the extracts to reduce the tendency to form emulsions. 11. Extract the HC1 solution with successive 25 ml por-tions of ethyl ether u n t i l no red fluorescence can be seen i n the ether extraction under uultraviolet light. Nor-mally two extractions are sufficient. Collect the ether extractS'-in a separatory funnel. 12. Wash the ether extracts with 5 ml of 1% Na2C03. Dis-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 in a graduated centrifuge tube. 15. Check the second extract with an ultraviolet light 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 slightly elevated protoporphyrin levels a l l fluorescing substances w i l l be removed in the f i r s t 25% HC1 extraction. If fluorescence persists, continue to extract with 0.5 to 1.0 ml quantities of 25% HC1 unti l the fluore-scent material is 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 con-centration. If Congenital erythropoietic porphyria or Erythroheptic copropor-phyria are suspect and the composition of the blood porphyrins i s de-sired step 5 of the above procedure i s followed by esterification and 2 chromatography . Occasionally, this procedure i s augmented with aqueous HC1 extraction for the total quantitation of uroporphyrin^. 21 C. HPLC BACKGROUND 1. RESOLUTION THEORY Resolution is 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 specific chromato-28 28 29 graphic procedure . It is dependent on ' : Selectivity - a measure of relative compound retention Capacity - a measure of analysis time defined as k' = volume of sample elution (Ve) - void volume of column (Vo)/Vo Random dispersion - a measure of the efficiency of a column (peak band width) which is defined in terms of the height equivalent of the theoretical plate (H) such that H = A + Cu11. The theoretical plate i s defined as the column volume element of minimum dimension such that the partitioning 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 diffusion or non-homogeneous flow and is dependent on particle size. Cy11 is a measure of the non-equilibrium which results from resistance to mass transfer in the stationary and mobile phases. The smaller the value of H the more plates/meter generated and the greater the efficiency of resolution. This i s usually the most important factor in liquid chromatography and is the cause of the large effect on both stationary and mobile phase mass transfer seen with particle size reduc-tion. Additionally, H may be decreased by peak assymmetry which results from the effect of multiple mode mass transfer (co-occurance of several different adsorption/desorption processes). 22 The increased resolution which results from decreased particle size was the principle factor which lead to the development of high pressure 28 (speed) liquid chromatography . As the average particle size decreases analysis time increases unless the solvent is 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 variation of the forces attracting solutes to the adsorbent and the forces tending to remove them from the adsorbent so they w i l l move with the mo-b i l e phase. 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 is based not on adsorption/desorption processes but on solute molecular size separations by diffusion through a rig i d gel notework. The larger solute w i l l be more diffusion re s t r i c -ted and w i l l elute before smaller molecule solutes. Frequently, however, 28 adsorption processes occur with permeation processes in a gel separation 3. PORPHYRIN HPLC The application of HPLC to the analysis of ; naturally occurring 30 porphyrins was unknown before this work was initiated . Subsequently, in 23 addition to the procedures reported here ' , techniques have been 17 33 developed for the analysis of uro to proto porphyrins ' and the separation of harderoporphyrin/isoharderoporphyrin 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 direct probe insertion 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 for 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 stainless steel tubing) placed on the valve and loop injector. VALVE/LOOP INJECTOR COLUMN PUMP SOLVENT SEPTUM INJECTOR 254 nm DETECTOR F L O W 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 for a Cary 17 spec-trophotometer. This c e l l allows detection in the uv - vi s i b l e and stop-flow uv - v i s i b l e scans. The sample volume i s ca. 60 u l . FIGURE 5. DIAGRAM OF THE HPLC/CARY 17 FLOW CELL. The quartz windows are symmetrically disposed, only the right-hand one has been sectioned i n the drawing. The body dimensions are 2.5 x 2.5 cm and the solvent beam path i s 2 x 7.4 mm (quartz to quartz). i 26 B. SOLVENT AND COLUMN PREPARATION 1. SOLVENTS Methanol and acetonitrile were spectro quality. Ammonium hydrox-ide, n-propanol, triethylamine (TEA), ethyl acetate, ethyl propanoate and propyl acetate were reagent grade. Propyl propanoate was made by stirr i n g propionic acid in propanol/H^SO^ overnight followed by extraction and d i s t i l l a t i o n . The light petroleum (30-60°) and methylene dichloride were glass d i s t i l l e d and had 10 ul 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 equilibration with the desired, solvent mixture. 2. COLUMNS Polyamide, Corasil C-18, Neutral alumina, Basic alumina, Porasil C, Porasil T and Corasil II (Table VII) were dry packed in 2.0 mm x 60 cm stainless steel chromatography columns. Sephadex LH-20 (Table VII) was slurry packed with methanol/methylene dichloride (1/1) in 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 DESCRIPTION SOURCE Polyamide Corasil C-18 Neutral Alumina A iiylon type adsorbent bonded on an impermeable bead A linear 18-carbon alkane bonded to a s i l i c a coating on an impermeable bead. Particle size 37-50 ym. A neutral alumina with particle size 18-30 um. Reeve Angel Waters Assoc. Waters Assoc. Basic Alumina A basic alumina with particle size 18-30 urn.' Waters Assoc. Porasil C A spherical totally porous s i l i c a with particle size 37-75 ym. Waters Assoc, P o r a s i l T An irregular totally porous s i l i c a with particle size 25-37 um. Waters Assoc. Corasil II A s i l i c a coating on an impermeable bead with particle size 37-50 ym. Waters Assoc. Sephadex LH-20 A semi-rigid bead formed dextran gel. Pharmacia 28 C. PORPHYRIN IDENTIFICATION AND QUANTITATION PROCEDURES 1. PORPHYRIN STANDARDS The following samples were used as standards: Uroporphyrin III from copper uro III (Porphyrin products) demetallated with ^SO^ and esterified with BF^/methanol; Uroporphyrin I (Porphyrin products) esteri-fied with diazomethane; coproporphyrin I and III were samples found i n our lab; protoporphyrin IX (prepared by RK DiNello) esterified with BF^/ methanol. The hepta-, hexa- and penta- carboxylic acid porphyrins were 21 obtained by sealed tube degradation of uroporphyrin followed by diazo-methane esterification. The tricarboxylic acid porphyrin was isolated from porphyric urine samples. Copper protoporphyrin IX and copper copropor-3 6 phyrin III were made by the procedure of Doss . Hematoporphyrin (Nutritional biochemicals) was esterified with BF^/methanol. Ethyl and propyl esters were prepared by ethanol or propanol/BF^ esterification of the respective porphyrins. 2. STOP-FLOW VISIBLE SPECTRA Visible spectra of compounds eluted from the HPLC column were made by stopping the solvent flow when the sample was in 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 for direct probe insertion 29 4. QUANTITATION Standard samples of uroporphyrin and protoporphyrin were used to calibrate microgram porphyrin/peak area response. Using the Cary 17 detection system at 403.5 nm, .2 absorbance units f u l l scale and 10.4 mm/ min. chart speed a value of .0192 ± .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. F l o r i s i l (1 g, 100-200 mesh) was suspended in 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 settled, .celite (300 mg; the celite was previously washed with 20% HC1 to remove a yellow pigment) in d i s t i l l e d water (25 ml) was added to the column. A 10 ml urine sample was then carefully acidified with 25% hydrochloric acid (one f i f t h the volume of urine) and after a brief degassing on a water aspirator the sample was added to the column. The column was washed with successive 25-ml aliquots of 5% HC1, 0.1% HC1, 10% acetic acid: 95% ethanol (3:1; an additional volume of this 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% Tris (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 solution was cooled to 0°C and reacted with an ether-39 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 dis-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 anhy-drous sodium sulphate. The mixture was fil t e r e d and the f i l t r a t e taken to dryness on a rotary evaporator. The residue was dissolved i n methylene dichloride (0.50 ml) for analysis. 2. FECAL SAMPLES 20 ml of methanol and 2 ml of BF^'Et20 were added to the fecal 4 sample (.5 g wet or .2 g dry) . The solution was stirred overnight. The esterified material was extracted with two 100 ml portions of methylene dichloride after the addition of 80 ml of water. The methylene dichloride solution was dried over anhydrous sodium sulphate, fi l t e r e d and the solvent removed on a rotary evaporator. A sample of the residue was dissolved in methylene dichloride (1.0 ml/.10 g dry feces) and analysed by HPLC gel chromatography. The residue was dissolved in a minimum of solvent and chromatographed on 60 g of s i l i c a gel Woelm activiy IV in a 2.5 x 45 cm glass column. The solvent was benzene/ethyl acetate/methanol (40/10/2). 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 in methylene dichloride (1.0 ml/.10 g dry feces) for analysis. In addition to the standard fecal sample preparation procedure described above a large sample of porphyric feces (VGH - lb) which contained significant quantities of sub-uro material was prepared for analysis. 125 ml of methanol and 8 ml of BF 3'Et 20 were added to a 1.254 g (dry weight) fecal sample. The solution was stirred overnight and extrac-ted with 3 x 500 ml portions of methylene dichloride as described pre-viously.:. After measurement of the total Soret absorption the residue was dissolved in a minimum of solvent (as above) and chromatographed on 350 g of s i l i c a gel Woelm activity IV in a 5.6 x 60 cm glass column. The fractions were: Discard Uro to proto porphyrins Sub-uro porphyrins 400 ml 1055 ml 900 ml 32 After the fractions were measured for total Soret absorption the sub-uro residue was dissolved in a minimum of solvent and chromatographed on 180 g of s i l i c a gel Woelm activity IV in 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 total Soret absorption. E. SPECTROSCOPIC ANALYSIS OF THE SUB-URO FRACTION 100 ul of the sub-uro fraction obtained by column chromatography was diluted to 2.5 ml with methylene dichloride 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 similar 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 urea-Trition extraction procedure of Rimington 1 1. 33 F. HPLC OF CLINICAL SAMPLES 1. URINE SAMPLES Urine samples were analyzed on a Porasil T column using a change in solvent reservoir from light petroleum (30-60°; no TEA)/methylene dichloride (TEA)/n-propanol (175/100/1) to light petroleum (30-60°; no TEA)/methylene dichloride (TEA)/n-propanol (40/100/1) at 14 ml after sample injection. The flow rate was 1.5 ml/min. 2. FECAL SAMPLES a. URO TO PROTO PORPHYRINS The uro to proto porphyrin fraction was analyzed on a Corasil II column using the valve and loop injector to introduce a light petroleum (30-60°;TEA)/methylene dichloride (TEA) (40/100) solvent into a system pumping a light petroleum (30-60°;TEA)/methylene dichloride (TEA)/ n-propanol (15/100/.5) solvent. The flow rate was 1.0 ml/min and the sample was inj.ected 2.5 min after solvent "injection". b. SUB-URO PORPHYRINS The sub-uro porphyrin fraction was analyzed on a Corasil 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 (TLC) (methylene dichloride/ ethyl acetate/methanol (80/2/1): Rf; proto (.87), tricarboxylic acid porphyrin (.67), copro (.48)):of the red fluorescent band i n the tr i c a r -boxylic acid porphyrin position from VGH - 1 gave compound(s) with an aetio-type spectrum in methylene dichloride. Peak (nm) Relative absorption 398 19.7 501 1.00 533 .73 564.5 .66 622 .20 The mass spectrum of this isolate had major peaks at m/e 650 and 652. 2. COPPER COPROPORPHYRIN The TLC's in this 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 tricarboxylic acid porphyrin position of the HPLC analysis of fecal samples VGH - 2 gave a metalloporphyrin spectrum. 35 Peak (nm) Relative absorption 399 525 561.5 27.8 1.00 1.72 A sample of this compound was obtained by TLC (solvent A) collection of the pink,nan^fluorescent band at Rf = .71. This compound was de-metallated with H„S0, but not with HC1. Using solvent A TLC the metal I 4 free porphyrin had the same Rf (.55) as an authentic sample of copropor-phyria A synthetic sample of copper coproporphyrin had the same Rf in solvent A and the same vis i b l e spectrum i n methylene dichloride 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 in which a VGH-2 fecal sample and/or a free porphyrin were esterified with methanol/H^SO^ or methanol/BF^. After separation of the reaction mixture by solvent A or B TLC the relative concentrations were determined spectrophotometrically. H. IDENTIFICATION OF THE SUB-URO PORPHYRINS Hematoporphyrin IX and several HPLC and column chromatography iso-42 lated sub-uro porphyrin fractions were acetylated by known methods Attempts to obtain mass spectra of the acetylated products were largely un-successful. Hematoporphyrin IX gave an m/e for protoporphyrin IX. 43 These same samples were also sylilated 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 sylilated. reaction on the sub-uro isolates showed a significant decrease in Rf after reaction. 36 III. RESULTS A. INVESTIGATION OF HIGH PRESSURE LIQUID, CHROMATOGRAPHIC PARAMETERS FOR THE OPTIMIZATION OF PORPHYRIN ANALYSIS A variety 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 isocratic (one solvent com-bination) solvent system was extensively studied because application of isocratic elution to routine analysis is 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 yield a satisfactory system. The elution of protoporphyrin IX from polyamide was very slow with a l l of the solvent combinations tested which resulted in 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 acetonitrile/2% ammonium hydroxide. 37 -t-T 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, light petroleum (30-60°) and/or triethylamine in methylene dichloride, benzene or toluene. Basic alumina had poor selectivity and very broad peaks (Figure 7 ) . 38 1 1 1 1 1 4 2 4 2 TIME (min) FIGURE 7. PROTOPORPHYRIN IX DIMETHYL ESTER (2) and COPROPORPHYRIN II I TETRAMETHYL ESTER (4) ON A BASIC ALUMINA PACKING. Solvent: Methylene dichloride/n-propanol Neutral alumina had good s e l e c t i v i t y but t a i l e d too much to give acceptable r e s o l u t i o n (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 Porasil C had good selectivity but the range of packing particle size resulted in extremely broad peaks (Figure 9). 16 12 8 4 . | TIME (min) FIGURE 9. UROPORPHYRIN III OCTAMETHYL ESTER ON A PORASIL C PACKING. Solvent: Methylene dichloride(TEA)/n-propanol Corasil II and Porasil T systems gave good overall resolution. However, even though i t had been determined on neutral alumina that substitution of ethyl propanoate for ethyl acetate in a methylene dichloride/methanol system (Table VHI-a) or the use of ethyl or propyl esters (Table VHI-b) significantly reduced k' for the higher carboxy-lated porphyrins, the effect was not iarge enough to allow isocratic elution of a complex sample on either Corasil II or Porasil T. There-fore, a methylene dichloride/light petroleum (30-60)/n-propanol/triethylamine 40 TABLE VHI-a A COMPARISON OF THE EFFECT OF ETHYL ACETATE AND ETHYL PROPANOATE ON PORPHYRIN RETENTION (k') Sample Coproporphyrin Solvent Ethyl acetate k'(%) 2.0 (100) Ethyl propanoate k'(%) 2.0 (100) Pentacarboxylic acid Hexacarboxylic acid 3.1 (100) 5.2 (100) 2.9 (94) 4.6 (89) a. 1% of ester and .2% methanol in methylene dichloride TABLE V l l l - b A COMPARISON OF THE RELATIVE VARIATION IN RETENTION (k') WITH PROPYL, ETHYL AND METHYL PORPHYRIN ESTERIFICATION3 Porphyrin Protoporphyrin Coproporphyrin Ester Methyl k'(%) 1.8 (100) 5.5 (100) Ethyl k' (%) 1.7(94) 4.6(84) Propyl k'(%) 1.6(89) 3.5 (63) a. Solvent: .15% methanol in methylene dichloride 41 system was chosen because i t was found to be applicable to the simple step gradient illustrated by the 254 nm baseline i n figure 10. If an electronic programmer were available methylene dichloride/n-propanol (.3-1.0% n-propanol) would probably give a good chromatogram. However, for routine c l i n i c a l analysis the operationally simpler system seemed best. 35 30 I 1 — r — 25 20 15 time (min) 10 "T~ 5 0 FIGURE 10. ILLUSTRATION OF THE SOLVENT GRADIENT (254 nm). The spike at -2 minutes marks the change to lower polarity solvent. The sample i s injected at 0 minutes with the solvent front at 1.5 minutes. The higher polarity solvent begins to re-elute at 11 minutes. The peak at 20 minutes i s n-propanol/TEA which i s swept from the column by the lower polarity solvent. 42 Chromatograms produced by test samples on Corasil II and Porasil T. (Figures 11a and b) demonstrate the resolution obtained by HPLC in comparison to TLC or extraction techniques (see figure 3). An added ad-vantage of the HPLC procedure i s one step elution and quantitation which 44 can be further simplified by the use of an electronic processor . Note that Corasil II gives maximal resolution in the 2-4 carboxyl region and Porasil T gives maximal resolution i n the 4-8 carboxyl region. Corasil II is well suited to the analysis of fecal samples where the 2-4 carboxyl porphyrins dominate while Porasil T gives better resolution of the 4-8 carboxyl porphyrins which occur predominantly in urine. For the routine needs of a c l i n i c a l laboratory either column i s equally suitable. 1 1 1 1 20 15 10 5 TlME(min) FIGURE 11a. REFERENCE CHROMATOGRAM OF URO TO PROTO PORPHYRIN (8-2) METHYL ESTERS ON CORASIL II. 43 2 0 ~ 15 i5 5 T I M E ( m i n ) FIGURE l i b . REFERENCE CHROMATOGRAM OF URO TO PROTO PORPHYRIN (8-2) METHYL ESTERS ON PORASIL T. The retention time for 3 was determined separately. i i . GEL PERMEATION CHROMATOGRAPHY Chromatography with a Sephadex LH-20 gel gave f a i r resolution of a simple porphyrin mixture (Figure 12). To increase the resolution for analysis of a complex mixture would require increased analysis time and a small particle packing. Unfortunately, the available small particle 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 role i n por-46 phyrin gel chromatography . Therefore this system cannot be further developed. 44 19 17 15 13 T I M E ( m i n ) 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 for the chromatographic analysis of the sub-uro porphyrins on Corasil II. Both systems gave poor resolution as a result of broad peaks. See section III.C.2. c (P. 57) for representative chromatograms. 45 b. GEL PERMEATION CHROMATOGRAPHY Sephadex LH-20 with methylene dichloride/methanol did not significantly separate the sub-uro porphyrins as this heterogeneous group of compounds was too complex for significant resolution. See Section III.C.2.a (P. 50) for representative chromatograms. B. SAMPLE PREPARATION PROCEDURES 1. URINE SAMPLES The talc extraction procedure for urinary porphyrins as outlined in the introduction requires repetition of many inefficient steps to obtain reproducible results. Therefore, we modified a procedure based on f l o r i s i l chromatography recently developed by Schwartz for the separation of por-38 phyrins from other urinary components . This method does not require tedious repetition or long reaction times to ensure complete extraction 4 and esterification . After adsorption of the porphyrins from acidified urine and washing to remove non-porphyric contaminants the Schwartz method used buffers based on ammonium ion for porphyrin elution. However, we achieved better results in the diazomethane esterification step when an ethanolic Tris buffer was used. The esterification with diazomethane immediately con-verted a l l of the carboxyl groups to their methyl esters which avoided the time and yield problems inherent with overnight methanol/H^SO^ 4 esterification . The f u l l y esterified porphyrins were extracted from 'the 46 aqueous phase with ether and after solvent removal the sample was ready for HPLC analysis. Examination of the residual aqueous phase after porphyrin extraction, by excitation at 366 nm, showed no residual fluorescence. 2. FECAL SAMPLES The total analysis of porphyric fecal 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 esterification and TLC for the uro to proto porphyrins . This duplication of effort has been completely eliminated by combining the more efficient 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 fraction from the sub-uro porphyrin fraction. I n i t i a l l y the column was eluted with methylene dichloride/ethyl acetate/methanol but entrainment of uro to proto porphyrins in the sub-uro porphyrin fraction necessitated the use of a benzene/ethyl acetate/methanol system. The benzene/ethyl acetate/methanol column fractionation of the fecal porphyrins results i n the loss of a significant portion of the sub-uro material through irreversible adsorption to the s i l i c a gel. The data 47 obtained from the large VGH-lb sample indicates t h i s loss (based on the t o t a l Soret absorption of the o r i g i n a l extract) to be $5%. However, subsequent analysis of the methanol/BF^ extract by gel chromatography (section III.C.2.a.,P. 50) and extractive analysis of selected f e c a l samples by the Rimington method 1 1 (section I I I . C . 2 . c . i . , P.57) has shown that the eluted f r a c t i o n i s representative with respect to 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 difference) 48 C. SAMPLE ANALYSIS 1. URINE SAMPLES The res u l t s of the f l o r i s i l / P o r a s i l T analysis of 5 urine samples are presented i n figures 13a and b and table IX. T I M E ( m i n ) FIGURE 13a. CHROMATOGRAM OF URINE SAMPLE 5 (TABLE IX). 48 T I M E ( m i n ) FIGURE 13b. CHROMATOGRAM OF URINE SAMPLE 4 (TABLE IX). TABLE IX RESULTS OF THE HPLC ANALYSIS OF PORPHYRIC URINE Porphyrin analysis: Microgram/litre of urine (relative percent) Sample Number of carboxyl groups 3 4 5 6 7 8 1 50 (12) 190 (45) 180 (43) 2 24 (4) 60 (10) 230 (39) 270 (47) 3 190 (2) 5,600 (48) 1,290 (ID 60 (1) 440 (4) 2,950 (34) 4 25 (1) 2,150 (40) 660 (12) 50 (1) 280 (5) 2,220 (41) 5 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 results with the general characteristics out-lined in table V and the literature values presented in figure 14 indicate 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 (3 samples) HPLC (2 samples) 5-3% 3-2% 6-9% 4-44% 7-39% 5-11% 8-49% 6-1% 7-4% 5 6 7 8 8-38% 3 4 5 6 7 8 CHU47 (7 samples) 48 CARDINAL (2 samples) 4-2% 4-32% 5-3% 5-8% 6-3% 6-1% 7-23% 7-1% h 8-67% 4 5 6 7 8 8-50% 4 5 6 7 8 FIGURE 14. COMPARISON OF THE HPLC AND LITERATURE ANALYSIS OF SYMPTOMATIC AND CONGENITAL ERYTHROPOIETIC PORPHYRIA SAMPLES. The bar graphs represent relative per cent of the total porphyrin sample of the uro to tricarboxylic acid porphyrins (8-3). 50 Also, the HPLC method Is preferable:: to the standard techniques because i t is simpler to use once the system is set up, requires much less direct technician time, is reproducible by different 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 after methanol/BF^ extraction/esterification did not give quantitative (baseline) separation of the uro to proto porphyrins or the sub-uro porphyrins. However, the chromatograms obtained (Figure 15) can be used to determine the relative quantities of the proto, copro and most importantly the sub-uro porphyrins at a glance. 51 P r o t o V G H - 1 a V G H - 2 a S u b - u r o C o p r o SA-b + 2 0 1 5 T I M E ( m i n ) 1 0 FIGURE 15. HPLC GEL CHR0MAT0GRAMS OF SELECTED FECAL METHYLENE DICHLORIDE EXTRACTS. 50 ul of each sample injected. 400 nm absorbance to scale. 52 The greatly increased sub-uro region of the VGH-la sample chroma-togram with respect to the VGH-2a and SA-b samples substantiates the use of the column chromatography sub-uro fraction to qualitatively de-termine 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 in sub-uro excretion associated with Variegate porphyria does not represent de novo synthesis but instead is merely an increased accumulation and excretion of porphyrins present in other samples. If the small quantity of sub-uro material observed in the VGH-2a and SA-b samples had been of significantly different origin (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 fecal samples from 3 porphyric patients are presented in figure 16 and table X. —i : 1 1 r 20 15 . . . N 10 5 t ime (mm) 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 TO PROTO PORPHYRINS Porphyrin analysis: microgram/gram dry weight feces (relative per cent). Sample Number of Carboxyl Groups 2 3 4. 5 6 7 8 VGH-la 820 (30) 290 (10) 1340 (49) 61 (2) 140 (5) 42 (2) 52 (2) VGH-2a 77 (6) 330 (24) a 950 (68) 26 (2) P ( D b VGH-2b 180 (8) 340 (15) a 1650 (75) P (0) 25 (1) SA-a 850 (76) 83 (7) 66 (6) P (2) 43 (4) 47 (4) P (1) SA-b 770 (72) 105 (10) 93 (9) 34 (3) 49 (5) P (1) a. Copper Coproporphyrin b. Present (10-20 yg/g) Analysis of the porphyrins in the tricarboxylic acid porphyrin region of samples VGH-1 and VGH-2 by HPLC/stop-flow visible spectroscopy proved that they were different compounds and provided on example of the analytical capability and v e r s a t i l i t y of the HPLC/Cary 17 system. The aetio porphyrin spectrum compound of VGH-1 was found by mass spectral analysis to be harderoporphyrin and/or isoharderoporphyrin and related products from microbial activity in 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 esterification/extraction procedure either from 33 extraneously introduced copper or from copper present in the feces but not combined with coproporphyrin. TABLE XI COPPER COPROPORPHYRIN TEST SAMPLES Sample Esterification TLCa Result b Feces MeOH/BF3 A Cu-Copro (+) Feces Me0H/HoS0, z 4 A Cu-Copro (+) Copro Me0H/BF3 A Cu-Copro (-) Proto Me0H/BF3 B Cu-Proto (-) Feces + Copro MeOH/BF^ A Cu-Copro (+) C Feces + Proto MeOH/BF. A Cu-Copro (+) B Cu-Proto (-) a. Copper protoporphyrin and copper coproporphyrin had Rf. .35 and .17 respectively in solvent B. b. (+) present: copper coproporphyrin Soret ca. 25% of coproporphyrin Soret; (-) absent: copper coproporphyrin Soret « 1% of coproporphyrin Soret. c. Greatly reduced relative to the total coproporphyrin. 56 2 48 A review of the literature ' revealed that an unidentified copper porphyrin and/or a pink,non^fluorescent band has been observed in the appropriate position in the TLC analysis of urine and fecal samples 2 (especially those samples from Hereditary coproporphyrics ). The biolo-13 27 gical role of this compound is unknown ' although i t i s probably excreted 27 to the feces via the bile The VGH samples were obtained locally so a comparison of the 27 c l i n i c a l 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 results agree very well (sample VGH-2a) when Cli n i c a l HPLC analysis analysis .:. Protoporphyrin (yg/g) 74 77 Coproporphyrin (yg/g) 1270 1280 the values for copper coproporphyrin and coproporphyrin are combined. The other VGH patient (VGH-1) was diagnosed as a Variegate porphyric without extensive porphyrin analysis. 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 this sample represents a case of Erythrohepatic protoporphyria. 57 C. SUB-URO PORPHYRINS i . SCREENING PROCEDURES The sub-uro fraction obtained from the column chromatographic pre-paration of porphyric fecal samples was used to qualitatively determine the increase in the sub-uro porphyrins by measuring the corrected Soret absorption of the sample. The relative difference between corrected Soret absorptions (Table XII), the values obtained by the Rimington method (Table XII) and the gel chromatographic data presented previously (Section III.C.2.a, P.50) i l l u s t r a t e that the column procedure, which in one step yields the uro to proto porphyrins and the sub-uro fraction, can be utilized for screening sub-uro porphyrin levels. 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 .10 140 SA-a .11 SA-b .09 94 The VGH-1 and 2 results correlate well with table V and the work of 13 49 Rimington ... and Elder et a l which indicate a greatly increased level of sub-uro porphyrins for Variegate porphyria and a moderately increased;level for Hereditary coproporphyria. 58 i i . CHROMATOGRAPHY AND IDENTIFICATION Analysis of the sub-uro porphyrin column fraction by HPLC provided additional information on the composition and source of the sub-uro group. Although the chromatograms (Figures 17 and 18) represent only ca. 15% 8 4 T IME(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 total sub-uro porphyrins originally present in the methylene dichloride extract they i l l u s t r a t e that the fraction obtained from samples with large quantities of the sub-uro porphyrins i s of essentially -\ : h 8 4 T IME(min) FIGURE 18. CHROMATOGRAPHIC 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. The sub-uro A, B fractions obtained from the large- sub-uro porphyrin extraction were:used as standards to prove that porphyrins A and B on the chromatograms were different compounds. Porphyrin A is present in the 1.50% methanol in methylene dichloride chromatogram as the solvent front shoulder on porphyrinoB. Additionally, figure 19 demonstrates the use of a variable wave-length and scanning HPLC detector to determine the porphyrin composition of a complex sample. Porphyrin A is the only porphyrin of the 5 peaks recorded in the 254 nm spectrum. Various sub-uro porphyrin samples were acetylated and/or sylilated for further chromatographic and structure analysis. The acetylated samples exhibited an increase in chromatographic mobility (TLC and HPLC) for some compounds indicating the presence of reactive hydroxyls but the samples were not volatile enough for massr: spectral analysis. The sjl y l a t i o n reaction formed products which decreased substantially in TLC Rf. Therefore mass spectra were not attempted. 61 S T O P - F L O W SCAN (nm) FIGURE 19. 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 esterification/extraction procedures and HPLC packing and solvent;systems for the analysis of porphyrins from porphyric urine and fecal samples has led 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 for c l i n i c a l and biochemical analysis. The standard techniques are tedious 27 and inefficient which results in unnecessary 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 effici e n t l y provide analyticallresults for 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 fecal sample. In contrast, her. .clinical laboratory b i l l was in 27 excess of $6,000 . The a b i l i t y of the HPLC/Cary 17 system to identify unusual components was demonstrated by the characterization of the TLC similar hardero-type and copper coproporphyria. The standard techniques currently in use also have the distinct disadvantage of requiring a separate procedure for each type of sample. Besides the demonstrated fecal preparation the use of methanol/BF^ should provide extracts suitable for HPLC analysis from such diverse sources.'ias blood, liver and cultured tissue. Also, the experience gained from 17 35 the HPLC optimization analysis in combination with recent reports ' on 63 superior porphyrin resolution with microparticulate packings (5-10um) should allow the development of a single, convenient isocratic HPLC analytical procedure. Perhaps the most significant proofs of the advantage of the pro-cedures 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 joint clinical,biochemical and chemical team to u t i l i z e HPLC analysis of porphyric tissue cultures for deter-mination 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 fecal sub-uro porphyrins has been shown to be of use in the diagnosis of some porphyrias 1 1. Unfortunately, the procedures presently available for their determination are too involved to be of 27 much use in a c l i n i c a l laboratory and have been unable to give more than qualitative 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 for the c l i n i c a l analysis of this group. Although neither procedure currently provides quantitative information the easily obtained data has been shown to be suitable for the determination of relative sub-uro porphyrin increase. 64 The most significant aspect of this work with respect to research on heme metabolism as well as porphyria has been our prelminary success in the separation and identification of the protein porphyrin complex/ sub-uro porphyrins. Although l i t t l e exact structural information was obtained we found qualitative similarities in the content of samples from different sources. The application of recently reported techniques for the use of f i e l d desorption mass spectroscopy"^ in combination with an evaluation of ethy, propyl and possibly t-butyl esterification, acetylation and gradient HPLC elution on microparticulate packings should allow the analysis of not only a much larger portion of the sub-uro group but also improved resolution and analysis of structure. 65-BIBLIOGRAPHY 1. H. ELDER, CH. GRAY and D.C. NICHOLSON, J. Clin. Path., 25, 1013 (1972). 2. T.C. CHU and E.J.H. CHU, Clin. Chem., 13, 371 (1967). 3. L. EALES in "Current diagnosis 4", H.F. Conn and R.B. Conn, Eds., W.B. Saunders Co., New York, N.Y., 1974, p. 661. 4. Y. GROSSER and L. EALES, S.A. Med. J., 47, 2162 (1973). 5. G. HOLT I, C. RIMINGTON, B. TATE and G. THOMAS, Quart. J. Med., 2]_, 1 (1958). 6. P.R. BROWN, "High Pressure Liquid Chromatography; Biochemical and Biomedical Applications", Academic Press, New York, N.Y., 1973. 7. A.D. ADLER, Ed., "The Biological Role of Porphyrins and Related Structures", Annals of the New York Academy of Sciences, Volume 244, New York, N.Y., 1975. 8. A.H. JACKSON, H.A. SANCOVICH, A.M. FERRAMOLA, N. EVANS, D.E. GAMES, S.A. MATUN, G.H. ELDER and S.G. SMITH, Phil. Trans. R. Soc. Lond. 8, 273, 181 (1976). 9. G.H. ELDER, J. Clin. Path., 28, 601 (1975). 10. L. EALES, Y. GROSSER and W.G. SEARS in "The Biological Role of Porphyrins and Related Structures", A.D. Adler, Ed., Annals of the New York Academy of Sciences, Volume 244, New York, N.Y., 1975, p. 441. 11. C. RIMINGTON, W.H. L0CKW00D and R.V. BELCHER, Clin. Sci., 35, 211 (1968). 12. R.V. BELCHER, S.G. SMITH and R.F. MAHLER, Enzyme, 17, 53 (1974). 66 13. R. POULSEN, Personal Communication. 14. M.S. STOLL and C.H. GRAY, Biochem. J., 117, 271 (1970). 15. R. SCHMID and A.F. McDONAGH in "The Porphyrins", D. Dolphin, Ed., Academic Press, New York, N.Y., in press. 16. H. PLIENINGER in "The Porphyrins", D. 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W.I. WHITE, R.C. BACHMAN and B.F. BURNHAM, in "The Porphyrins", D. Dolphin, Ed., Academic Press, New York, N.Y., in press. 31. R.E. CARLSON and D. DOLPHIN, First Int. Porphyrin Meeting: Porphyrins in Human Diseases, Freiburg, 1975, i n press. 32. R.E. CARLSON and D. DOLPHIN in "High Pressure Liquid Chromatography in C l i n i c a l Chemistry", P.F. Dixon, CH. Gray, G.K. Lim and M.S. Sto l l , Eds., Academic Press, San Francisco, CA., 1976, p. 87. 33. N. EVANS, A.H. JACKSON, S.A. MATLIN and R. TOWILC i n "High Pressure Liquid Chromatography in Cli n i c a l Chemistry", P.F. Dixori, CH. Gray, CR. Lim and M.S. S t o l l , Eds., Academic Press, San Francisco, CA., 1976, p. .7.1. 34. J.A.S. CAVALEIRO, G.W. KENNERkand K.M. SMITH, J.C.S. Perkin I, 1189 (1974). 35. A.R. BATTERSBY, D.G. BUCKLEY, C C HODGSON, R.E. MARKWELL and E. McDONAID in "High Pressure Liquid Chromatography in Cl i n i c a l Chemistry", P.F. Dixon, CH. Gray, CK. Lim and M.S. St o l l , Eds., Academic Press, San Francisco, Ca., 1976, p. 63. 36. M. DOSS, Anal. Biochem., 39, 7 (1971). 37. J. MOLLER and T.K. WITH, Org. Mass. Spec, 9_, 443 (1974). 38. S. SCHWARTZ, Personal Communication. 39. M. FIESER and L.F. FIESER, "Reagents for Organic Synthesis", Vol. 2, Wiley, New York, N.Y., 1969, p. 102. 40. C. RIMINGTON and S.L. SVEINSSON, Scand. J. Clin, and Lab. Invest., 2, 209 (1950). 41. T.K. WITH, Scand. J. Clin, and Lab. Invest., ]_, 193 (1955). 68 42. Pyridine/Acetic Anhydride (9/1). 43. J.R. CHAPMAN and G.H. ELDER, Org. Mass Spec, b_, 991 (1972). 44. Hewlett Packard - 1080 Series High Pressure Liquid Chromatographs, Information Bulletin (1976). 45. WATERS ASSOC., Gel Chromatography Packings Technical Sheet. 46. R.C. BACHMANN and B.F. BURNHAM, J. Chrom., 41, 284 (1969). 47. T.C. CHU and E.J.H. CHU, J. Biol. Chem., 234, 2741 (1959). 48. R.A. CARDINAL, I. BOSSENMAIER, Z.J. PETRYKA, L. JOHNSON and C.J. WATSON, J. Chrom., 38, 100 (1968). 49. G.H. ELDER, I.A. 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