Here’s what Alan D. Adler could (should?) have done to investigate the porphyrins of those anomalous Shroud bloodstains.

Posted a minute ago to Dan Porter’s shroudstory.com site. It should be self-explanatory (I hope so since I’m busy with other matters for the next day or two with only occasional internet access). Hopefully the visual aids will be of some use  those whose knowledge of biochemical analysis is limited.

November 11, 2013 at 4:14 am | #61

As I say, Kelly, if you want to extract degraded or even minimally modified biomolecules of relatively small molecular weight, e.g. porphyrins or bile acids from what loosely might be described as gunk, you do not seek the services of a synthetic organic chemist, even if he is a porphyrin specialist. Why not? Because he’s not au fait with the problems of dealing with the biological milieu of proteins, lipids etc, of learning the little tricks that are needed to break attachments between your target molecule and the surrounding gunk, and then persuading that molecule, once free, to partition into an organic layer from which it can then be pipetted off and spotted straight onto a TLC plate and run against authentic standards.

My first years in research were spent having to acquire precisely these kinds of techniques, and had I encountered Alan Adler at coffee, shaking his head over his “ancient blood” problem, I’d have wasted no time in suggesting a certain fix, one I learned from the bile acid literature, and had successfully applied to bilirubin, and not just free relatively apolar bilirubin but its more polar conjugates (mono- and di-glucuronides).

What one does is exploit the fact that porphyrins and linear tetrapyrroles that originate from haemoglobin all have two propionic acid side chains that make them negatively charged organic anions, or partially anionic, in physiological buffers around about neutral pH. The trick is this. There’s a cationic surfactant called CTAB, cetyltrimethylammonium bromide, i.e. hexadecyltrimethylammonium bromide, which you may have heard of, since it’s used to extract DNA from chloroplasts and other plant material.

This is the structural formula for cetyltrimethylammonium bromide. Note the positive charge on the nitrogen, and the entirely separate negative bromide ion, needed for electrical neutrality, but inert in general usage,

This is the structural formula for cetyltrimethylammonium bromide. Note the positive charge on the nitrogen, and the entirely separate negative bromide ion, Br-, needed for electrical neutrality, but inert in general usage. The molecule is shown folded (probably to conserve space on its website.

This is a simple and more informative view of CTAB, with its positive nitrogen (blue), its negative bromide ion (red) and the long extended hydrocarbon tail (grey) rwithout which it would not possess detergent/surfactant properties.

This is a simple and more informative view of CTAB, with its positive nitrogen (blue), its negative bromide ion (red) and the long extended hydrocarbon tail (grey) without which it would not possess detergent/surfactant properties.

You add CTAB, and being cationic, it forms a tight association with you anionic porphyrins etc. You then add chloroform, methanol and buffer in the correct proportions to get two immiscible layers, i.e. a standard Folch extraction, and hey presto your CTAB-porphyrin associations  migrate from the aqueous to the organic layer.

Folch extraction. The porphrin-CTAB associations should migrate into the lower layer of the right hand diagram.

Folch extraction. Any ancient blood porphyrin-CTAB associations should migrate into the lower layer of the right hand diagram.

Take off that lower organic layer, maybe evaporate some surplus solvent with a puff of nitrogen, then as I say, run on TLC with a solvent of intermediate polarity, maybe with a dash of acetic acid to help split the CTAB-porphyrin associations.

Thin layer chromatography (tlc) on silica gel. The ascending solvent carries the different components at different speeds, allowing them to be taken off (eluted) as relatively pure substances for further analysis.

Thin layer chromatography (tlc) on silica gel. The ascending solvent carries the different components at different speeds, allowing them to be taken off (eluted) as relatively pure substances for further analysis.

All you need is a uv lamp to detect the porphyrins as they migrate up the silica gel. You can then take them off with one of those gizmos attached to a vacuum line whose name I have forgotten, and when the removed areas of silica gel have dried you tap out the powder, elute off your purified porphyrins etc for analysis by glc-mass spec or whatever.

I and Don Ostrow were using this technology in the early 70s to track the fate of bilirubin in the phototherapy of neonatal jaundice. Alan Hoffman at the Mayo Clinic  (lucky to be  alive after the recent mishap in his Toyota Prius) was doing the same thing to track the fate of bile acids in the enterohepatic circulation. Why on earth did STURP not take advice and tips from people dealing with biological material. Why did they recruit a synthetic organic chemist who, smart though he was, very smart by all accounts, was simply ill-equipped to deal with biological gunk?

Second comment, in response to  Kelly Kearse:

November 11, 2013 at 5:56 am | #63

Yes, but suppose his experiments using hydrazine to solubilize had fouled up (as indeed they may have) and suppose mine with the CTAB had failed to extract porphyrins into the organic layer. I can still confidently state that the porphyrins are still there, in their same chemical state, waiting for a different approach. Could Adler have said the same, given that hydrazine is a powerful chemical reagent, with the properties of both base and powerful reducing agent?

There’s a certain metaphorical sledgehammer that springs to mind, one that horrifies squirrels, including your rampaging greys. Hydrazine is a chemist’s sledgehammer – not what you need in a physiological milieu.STURP messed up on its recruitment.There should have been biomedical analysts looking at blood, not synthetic organic chemists or dare I say it, a thermochemist concerned primarily with explosives testing. I repeat: STURP fouled up – big time. The least it can do now, to make amends, is to publish all of its chemical and other analytical findings and protocols to the internet, open access.

I’ll try and look in later, but have grandfatherly duties to attend to for the next 36 hours or so.

Third comment:

November 11, 2013 at 5:56 am | #63

Yes, but suppose his experiments using hydrazine to solubilize had fouled up (as indeed they may have) and suppose mine with the CTAB had failed to extract porphyrins into the organic layer. I can still confidently state that the porphyrins are still there, in their same chemical state, waiting for a different approach.  Could Adler have said the same, given that hydrazine is a powerful chemical reagent, with the properties of both base and powerful reducing agent?

There’s a certain metaphorical sledgehammer that springs to mind, one that horrifies squirrels, including your rampaging greys. Hydrazine is a chemist’s sledgehammer – not what you need in a physiological milieu. STURP messed up on its recruitment.There should have been biomedical analysts looking at blood, not synthetic organic chemists or dare I say it, a thermochemist concerned primarily with explosives testing. I repeat: STURP fouled up – big time. The least it can do now, to make amends, is to publish all of its chemical and other analytical  findings and protocols to the internet, open access.

I’ll try and look in later, but have grandfatherly duties to attend to for the next 36 hours or so.

4th comment:

November 11, 2013 at 9:13 am | #67

Groan (ancient biochemical provenance): You make the business of identifying blood sound as simple as testing for iron oxide. Well,. that’s how the haemoglobin marker for blood  ends up eventually after centuries or millennia, but there is complex spectrum of changes along the way, each of which takes it further and further away from recognizable blood.

Just the changes in the first year alone can be progressive and subtle, requiring some 6 different techniques to monitor. See the recent paper by Bremmer et al (2012) “Forensic quest for age determination of blood stains”.

Repeat: “blood” is not something for which there’s a quick spot test, and you may need a battery of increasingly sophisticated techniques to be certain that an old stain used to be fresh human blood.

5th comment:

November 11, 2013 at 10:24 am | #69

Sadly, I would have to tell Alan Adler that he is not the person best equipped to pronounce on whether it is real blood or not – that so crucial a question should not have been left in so few hands, and that his resort to the bilirubin get-out to explain an anomalous spectrum was hardly in the best scientific traditions if linked to the biblical narrative, and a presumption of excess “traumatic” bilirubin. I would ask him to produce conclusive scientific evidence for the presence of any bilirubin in those bloodstains, much less an excess, pointing out gently that while he was known as a porphyrin specialist, (let’s avoid the e word, it  being highly context specific)  I was a bilirubin one, and not impressed by what I had seen thus far from his modus operandi.

Let’s not mince our words. The blood analysis was ill-thought out and frankly shambolic sub-optimal. There should have been initial experiments first on degraded blood of various ages, of various treatments designed to model advanced age, to find methods of retrieving the porphyrins etc in the native state. The deployment of ferocious agents like hydrazine was frankly incomprehensible. One simply does not do that in biochemistry and physiology.

Sixth comment

November 11, 2013 at 3:36 pm | #74

At the risk of seeming evasive (one man’s non-committal response is another man’s evasiveness) I think the problem is this – that a porphyrin specialist was recruited to investigate the bloodstains, but made no attempt to extract the presumed protoporphyrin IX that should have been there if real human blood. As I have indicated, the methodology was available to do that, provided sufficient blood scrapings had been taken the pigments to be tracked visually or spectrallyon solvent extraction and subsequent tlc against standards, maybe with mass spec confirmation. (But neither Adler nor Heller went to Turin with STURP, so had no direct say in sample collection sufficient for needs).

Why use the services of a porphyrin specialist who did not accompany the team to Turin, and who made no attempt at physical isolation of the porphyin, relying instead on peaks and troughs in uv/visible spectra, and then attempting to explain away an anomaly by positing the presence of “trauma” bilirubin, betraying a lack of strict scientific objectivity.  He was supposed to garner chemical evidence for authenticity – not assume authenticity as soon as the going got difficult.

So if you ask me whether it was real blood, I’m entitled to reply – “maybe, but not necessarily collected fresh from a crucified man with typical porphyrin  spectrum, mineral composition etc”. Other scenarios are possible that are consistent with the radiocarbon dating’s medieval providence (but I’ll spare you the L word, having been content in the past to show how alternative scenarios are possible that might, just might, explain the anomalies).

7th comment

November 12, 2013 at 8:24 am | #79

“… the characteristics of blood post-Leech, that is showing how blood might become atypical maybe blood….”

Yes, Kelly, there is actually. It’s called hydroxyproline (HP), a breakdown product of collagen and elastin in connective tissue. Given there’s a lot of connective tissue in a leech that may have been extruded along with semi-digested blood (depleted in potassium and with an anomalous porphyrin spectrum) by our Brother Hirudo, looking for a handy look-alike non-clotting substitute for fresh blood, there’s actually quite a few aspects that could be said to fit a ‘wacky’ leech hypothesis.

But jump forward to the late 20th century, where an explosives thermochemist recruited to STURP as Chemical Director detects a hydroxyproline peak on his pyrolysis mass spectrum of Shroud “blood” , and what’s his response? Answer: to assume it’s normal to have HP as a major constituent of human blood (where’s the evidence?) and immediately to seize upon it as a handy way of bolstering a flimsy case against the Shroud image being a scorch, claiming that the heat would have driven off HP (despite it being a relatively-involatile high melting solid which he probably detected via its fragmentation pattern rather than improbably volatile molecular ion).

But there’s a long, long new posting that has just gone up on this site that has confirmed my long-held suspicions that anything one says about the hallowed Raymond Rogers and his peculiar blind spots in chemistry will be totally ignored, like my recent dismissal of his claims that thymol makes further radiocarbon dating a waste of time (bless!). I even provided an email address for the American Chemical Society, inviting folk here to get a second (and better-informed) opinion on what I consider to be generic eye wash rather than branded mouth wash. Yet all his outrageous claims that thymol would form chemical bonds with the cellulose of linen have been totally ignored, and his phoney chemistry endorsed as if unchallengeable fact. I am clearly wasting my time here.

Anyone who claims (like John Klotz) that I am a poodle barking at Great Danes could do a lot worse than consult Google Scholar, and check my credentials against those of Shroudie Land celebs (enter R.N.Rogers, A.D.Adler, J.H.Heller etc and compare with mine – C.S.Berry).

Thank you for the putdown, John Klotz. I had never thought of using Google Scholar to check names instead of topics until you made that poodle remark. I see no further point in being modest about my contributions to biomedical science when there are folk like you on the site, attempting to turn Shroudology into a scientific beauty contest, one in which the biggest pair of tits (fake as often as not ) make the biggest impression on the panel of self-appointed judges.

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About Colin Berry

Retired science bod, previous research interests: phototherapy of neonatal jaundice, membrane influences on microsomal UDP-glucuronyltransferase, defective bilirubin and xenobiotic conjugation and hepatic excretion, dietary fibre and resistant starch.
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