Diagnosis of diabetes mellitus at the Hospital of Venice in 1863

Clinica Chimica Acta 297 (2000) 17–27www.elsevier.com/ locate /clinchim

Diagnosis of diabetes mellitus at the Hospital ofqVenice in 1863

a b ,*Giuliano Dall’Olio , Romolo M. DorizziaClinical Chemistry and Haematology Laboratory, Ospedale S. Bortolo, via Rodolfi 4, 36100 Vicenza,

ItalybClinical Chemistry and Haematology Laboratory, Azienda Ospedaliera di Verona, Piazzale Stefani 1,

37126 Verona, Italy

Received 18 September 1999; received in revised form 3 January 2000; accepted 10 February 2000

Abstract

In 1674 Thomas Willis reported that the presence of urine ‘as sweet as honey’ was thepathognomonic sign of diabetes mellitus. In the 19th Century several reactions for the detection ofglucose in urine were proposed and glucose measurement became common in the laboratories thatwere being set up in Europe. A case of diabetes mellitus, diagnosed by Namias, the head of theWomen’s Section of the Medicine Department of Venice Hospital, was reported in 1863 in the‘Giornale Veneto di Scienze Mediche’ which contains clinical and laboratory information. A34-year-old woman was admitted to the hospital for polydypsia, polyuria, bulimia and fatigue.

21Urine was weighed for 2 months (2–10 kg day ) and the relative density ranged from 1.045 to1.038. Glucose was measured in the urine using Moore, Trommer and Fehling reagents. A fewdays after admission a urine sample showed 7.69 parts /100 parts of urine and a blood sampleshowed 547 mg of glucose /100 g of serum. The assays were carried out in the Clinical Laboratoryof Venice Hospital, founded in 1863, directed by Giovanni Bizio, one of the first chemists whograduated at Padua University. In 1863 chemical analyses were commonly carried out in Venice asin the other parts of Habsburg empire. 2000 Elsevier Science B.V. All rights reserved.

Keywords: History; Diabetes mellitus; Glucose; Venice; Giovanni Bizio; Giacinto Namias

qPresented in part at the Uroscopy to Molecular Analysis (Improving Diagnostics from Urinalysis) Meeting,Kloster Seeon, Bavaria, Germany, 18–20 September, 1999.

*Corresponding author. Tel.: 1 39-45-807-3248; fax: 1 39-45-807-2156.E-mail address: [email protected] (R.M. Dorizzi)

0009-8981/00/$ – see front matter 2000 Elsevier Science B.V. All rights reserved.PI I : S0009-8981( 00 )00230-8

18 G. Dall’Olio, R.M. Dorizzi / Clinica Chimica Acta 297 (2000) 17 –27

1. Introduction

In 1674 Thomas Willis (1621–1675), professor of physiology at Oxford,reported that the presence of urine ‘as sweet as honey’ was the pathognomonicsign of diabetes mellitus. In 1776 Matthew Dobson (1735–1784) demonstratedthat the sweetness of the urine of diabetic patients was caused by the presence of

`sugar and in 1825 the French chemist Michel Eugene Chevreul (1786–1889)classified it as glucose (grape sugar). In 1835 the Italian chemist FeliceAmbrosioni (1790–1843) found glucose in the venous blood of a diabeticpatient confirming the hyperglycemia report of John Rollo (1797) and the

`hypothesis of Nicholas Vaquelin (1825) and Eugene Souberian (1826). ClaudeBernard (1813–1878), investigating digestion, clarified the glycogenic role ofthe liver [1,2].

These experimental observations supported the idea that laboratory examina-tions could be used as a ‘chemical sign’ of illness as first proposed in 1827 byRichard Bright (1789–1858) who indicated albuminuria as the characteristicsign of the disease that would be named after him [3]. According to LudwigThudichum (1829–1901), one of the fathers of clinical chemistry, the results ofchemical examinations were useful in the ‘chemical identification of thedisease’, i.e. in the diagnosis [4]. The interest for diagnosis-oriented chemicalinvestigation and for the chemical methods started to grow more and more. Newand more sensitive reactions for the detection of glucose in biological fluids(especially urine) were proposed in 1841 by Carl August Trommer (1806–1879), in 1844 by Charles Louis Arthur Barreswil (1817–1870) and in 1844 byHermann Fehling (1812–1885). In the second half of the century many othertests were proposed (e.g. the reaction of fermentation and the reactions proposed

¨by Moore, Worm-Muller, Bottger, Nylander, Primavera, Mulder, Johnson,Penzoldt, Rubner, Molisch, Luton and, around 1890, the easy-to use strip test)[5–7].

In 1840 the French optician Jan Baptiste Soleil (1798–1878) developed thepolarimeter which was used very soon for measuring urinary glucose in clinicallaboratories.

2. The case report

A case of diabetes mellitus, diagnosed by Giacinto Namias, the head of theWomen’s Section of the Medicine Department of Venice Hospital, was reportedin the ‘Giornale Veneto di Scienze Mediche’ (Italian Journal of the VenetianMedical Sciences) in 1864 which contains clinical and, especially, laboratoryinformation [8] (Fig. 1).

G. Dall’Olio, R.M. Dorizzi / Clinica Chimica Acta 297 (2000) 17 –27 19

Fig. 1. First page of the referred article reporting a case of diabetes mellitus published in the‘Giornale Veneto di Scienze Mediche’ [8].

2.1. History

A 34-year-old woman, who worked as a greengrocer, was admitted to theGeneral Hospital of Venice on 20th August 1863. The disease appeared after 14months of nursing her child. In the past she had suffered from quartan andtertian fever and an enlarged spleen. After 2 months, she consulted a generalpractitioner and, only after the symptoms had become severe, was she admittedto the hospital. Her symptoms were a very strong thirst that could not besatisfied (polydypsia), polyuria, bulimia and general fatigue.

Since diabetes mellitus was strongly suspected, the clinician requestedchemical investigations and an examination by the eye specialist since eyecomplications from the illness were already known [1].

The examinations demonstrated the presence of glucose in the urine confirm-ing the diagnosis of diabetes mellitus.


2.2. Etiology

The disease was connected to malaria fevers, nursing for too long, emotionalstress and extreme poverty.

2.3. Treatment

Her drinking was limited and her diet was restricted to a little bread and meatmixed with sugar. Then diarrhea and abdominal pain started; therefore bismuthnitrate, sodium bicarbonate and opium were prescribed. In this phase glucosewas measured several times in the urine. After 8 days the patient began to sufferfrom throat pain and difficulties in swallowing and glucose was measured also inthe blood. At the end of September, when the abdominal pain persisted, atreatment of cod oil, drops of ‘tintura tebaica’ (based on opium) and iron sulfatewas started and the general condition improved (less glucose could be found inurine). On the 12th of November, the general conditions worsened: fever, diffusepain, headache, difficulties in swallowing appeared, followed by smallpox signs(coupled to polydypsia and polyuria lessening, disappearance of glucose and theappearance of albumin in the urine). The patient died after a few days.

2.4. Post-mortem examination

A post-mortem examination was carried out 30 h after the death: the floor ofthe fourth ventricle of the brain was hardened, the liver was enlarged and wasfull of very dark blood in enlarged vessels, the spleen was normal, the kidneyswere enlarged and full of blood.

2.5. Final report of the attending physician

After 8 months of disease, the patient died from smallpox not from diabeteswhich was treated using the available symptomatic therapies since the cause ofhyperglycemia in diabetes was as yet unknown.

Some previous observations were confirmed: sugar disappeared and albuminappeared in the final phase of the disease; the post-mortem finding of thehardened fourth ventricle floor was consistent with Claude Bernard’s experi-ments (1849), demonstrating glucose in the urine of rabbits and dogs afterdamage in the fourth ventricle floor. The enlargement of the liver and kidneyscould be explained, respectively, by the large amount of glucose that must bemetabolized and by the large amount of water eliminated in the illness.


2.6. Laboratory tests

The weight of urine was measured for 2 months after the admission (range:212–10 kg day ) and the relative density ranged from 1.045 to 1.038.

Glucose was measured in the urine using the common reagents (Moore,Trommer, Fehling). A urine sample analyzed after a few days showed: relative

¨density 5 1.041, solid substances (Haser) 5 9.553 parts /100 parts of urine;glucose 5 7.69 parts /100 parts of urine. A 16.45-g sample of blood was sent toProf. Bizio for the measurement of glucose (Fehling method) which showed 547mg/100 g of serum.

2.7. The analysts

Around the middle of the 19th Century the simplest analyses were carried outby the physicians themselves at the bedside. The most sophisticated analyseswere entrusted to other professionals with a better knowledge of analyticalchemistry, the apothecaries, who worked in the major hospitals charged with thepreparation of drugs.

Only after the institution of the Faculty of Chemistry in Italy (the first degreewas awarded at the University of Padua on the 13th of December 1838) [9],some chemists became available for carrying out chemical analyses. In themonths of the described case report, the head of the Women’s Section of VeniceGeneral Hospital was Giacinto Namias (1810–1874) (Fig. 2), a physician who,following the most modern trends in medicine, held the diagnostic usefulness ofchemical analyses in the highest esteem. He himself carried out simple analysesand sent the more complex ones to the apothecary of the hospital (PasqualeCappelletto) or to the chemist Giovanni Bizio. Bizio (Venice 1823–1891) (Fig.3), graduated in Chemistry at Padua University in 1847 and worked from 1858to 1862 at the Chemistry Laboratory of Vienna (Redtembacher, 1810–1870),Munich (Liebig) and Heidelberg (Bunsen), where he acquired a wide experiencein analytical chemistry. Back in Venice he taught chemistry and obtained publiccommissions. He coordinated the chemical, geological and thermal studies of themineral water springs in Veneto (1856–1878) [10]. In 1862 he was offered theChair of Organic Chemistry at the University of Naples but he did not accept it.Bizio was the first graduate in Chemistry to work in a clinical laboratory in Italy.From 1863 he taught at the ‘Scuola Pratica di Medicina’ (Practical School ofMedicine) and then was Chemist of Venice Hospital (Fig. 4). Moreover, Biziopioneered the studies of the effects of the matrix on the biological samples [11].He published several interesting studies of chemistry (more than 100 papers).Among the subjects investigated were the interferences of some components ofurine in some methods; a study on the presence of glycogen in invertebrateanimals, a method for detecting indacan in the sweat which would be reported in


Fig. 2. Giacinto Namias (1810–1874), head of the Women’s Section of the Medicine Departmentof Venice Hospital.

the Treatise of Physiological and Pathological Chemistry Analysis by FelixHoppe-Seyler (1870) [12,13].

3. Laboratory methods

3.1. Relative density

Relative density was measured using the pycnometer: a small bottle with aglass holed cap connected to a capillary tube with an impressed mark for easily


Fig. 3. Giovanni Bizio (1823–1891), chemist at the Venice Hospital.

Fig. 4. The Venice Hospital, 1847 (from [15], modified).


measuring the amount of the poured fluid. The pycnometer was weighed threetimes: first empty and dried; then full of water up to the mark and finally full ofurine up to the same mark. Subtracting the weight of the first from the secondand the third, the weight of the examined fluid and that of the same volume ofwater were obtained. Their ratio indicated the relative density.

Bizio reported, in the presented case, that the weight of the empty pycnometerwas 3.9312 g, the weight of the pycnometer full of water was 26.1245 g, theweight of the pycnometer full of urine was 27.0375 g and the relative density

¨was 1.041. He calculated, using the Haser formula [41 (the last digits of the21relative density) 3 2.33 5 95.53 g l ] the solid substances contained in a litre of

urine.

3.2. Glucose

The detection of glucose in the urine was a difficult procedure. In order toavoid mistakes, the apprentices were especially advised to practice measuringglucose in samples containing low and high concentrations of glucose (obtainedby adding the internal part of a fig or a grape to a urine sample).

3.2.1. Moore–Heller reactionTwenty millilitres of urine were poured into an evaporating dish; 2 ml of a

concentrated solution of potassium hydroxide were added and then the mixturewas boiled. The presence of glucose was revealed by the yellow colour in thebottom of the porcelain evaporating dish. The shade of the colour wasproportional to the concentration of glucose and when it was very high itdarkened up to the colour of black ink. When mucin was present, it was better totreat urine with lead acetate and filter it in order to obtain a clear fluid.

3.2.2. Trommer reaction (1841)One millilitre of a solution of potassium hydroxide and some drops of a 10%

solution of copper sulfate were added to 5 ml of urine and the mixture wasboiled. When glucose was present in the urine, the blue colour turned yellow andfinally red (copper oxide). Trommer was able, using this reaction, to detect sugarconcentrations down to 0.001% and 0.0001%. Other reducing substances presentin the urine, such as uric acid, creatinine, mucin and xanthine, could interfere inthe reaction [5–7].

3.2.3. Fehling reaction (1849)This was the classic method for detecting glucose based on its reducing power

of copper hydroxide employing two reagents. Five ml of reagent A (34.6 g ofcopper sulfate in 500 ml of water) and 5 ml of reagent B (175 g of Seignettesalt, sodium and potassium tartrate, and 50 g of sodium hydroxide in 500 ml of


water), the Fehling reagent, were reduced by 0.05 g of glucose. Ten ml of mixedFehling reagent was poured into an evaporating dish and diluted (1:10 or 1:20)urine into a burette. The dish was heated up to boiling point and then the dilutedurine was added, using the burette, to the reagent until the blue colourdisappeared.

Bizio used the following procedure: 50 ml of urine was mixed with 950 ml ofdistilled water. Ten ml of the mixed Fehling reagent was reduced by 12.5 ml of

21urine; therefore the concentration of glucose was 8 g dl . Since the relativedensity was 1.041, the result was, in percentage part /part, 7.69 g of glucose /100g urine.

3.2.4. Glucose in blood`Bizio explained ‘‘Per coagulare l’albumina, aggiunsi al siero una quantita di

alcool pari a 6 volte il suo volume. Spremuto bene il coagulo e reso il liquidolievemente alcalino con acido tartarico, distillai l’alcool ed evaporai a secchezza.Aggiunsi altro alcool all’80% per separare la maggior parte dei sali, filtrai lasoluzione alcoolica, evaporai a secco e portai il tutto a 50 ml con acqua. Per ladeterminazione quantitativa dello zucchero usai una soluzione normale alcalinadi solfato di rame (1 ml 5 0.005 g di zucchero). 10 ml di questa soluzionerichiesero, per la completa riduzione del rame, 31 ml delle soluzione acquosa delsiero’’ [8]. [I mixed 16.45 g of serum with alcohol (a volume 6 times greater) forcoagulating albumin; then I added tartaric acid, distilled the alcohol and broughtthe residue to dryness. I added alcohol for separating most salts, filtered thealcoholic solution, newly brought to dryness and again added 50 ml of water. 31ml of this aqueous solution were needed for reducing 10 ml of an alkalinesolution of copper sulfate.] Taking account of the dilutions, Bizio calculated aglucose concentration of 0.547 g in 100 g of serum.

3.3. Albumin in urine

3.3.1. Nitric acid reaction (qualitative)A few ml of urine were heated up to boiling point. Then a volume of diluted

(1:10–1:20) nitric acid (d 5 1.18) was added to the urine, producing aprecipitate. It was possible to discriminate albumin from a phosphate precipitateby adding more nitric acid; the albumin precipitate persisted while the phosphateprecipitate dissolved [5].

3.3.2. Acetic acid and potassium ferrocyanide (qualitative)Three to four millilitres of clear, filtered urine were mixed with the same

volume of acetic acid and a few drops of potassium ferrocyanide. A hugeprecipitate was formed when a large amount of albumin was present while aclouding or a pale opalescence appeared in the presence of a small quantity of


albumin. This reaction allowed the detection of concentrations of albumin as lowas 0.0008% [5].

3.3.3. Heller reaction (1852) (qualitative)A few ml of urine were poured into a tube and a drop of nitric acid was

slowly added along the wall of the tube. When albumin was present, a whitecloudy ring was formed which disappeared after shaking and appeared againafter the addition of another drop of acid [5].

3.3.4. Gravimetric quantitative measurementSixty to one hundred millilitres of urine was poured into a porcelain

evaporating dish; after bain-marie heating (heating a small vessel in a largervessel of hot water) a 2% solution of acetic acid was added drop by drop; in thepresence of albumin, a wooly precipitate was formed. After boiling, theprecipitate was transferred onto a filter of known weight and was washed withwater, alcohol and ether. The filter was dried at 120–1308C until it reached aconstant weight. The difference between the weight of the filter with theprecipitate and the filter alone was equal to the albumin contained in the urine[5].

4. Conclusions

The report of a case of diabetes mellitus, diagnosed and treated at VeniceHospital in 1863, published in one of the most important Italian medical journalsdemonstrates the role of the clinical chemistry laboratory in an importanthospital of the Habsburg Empire (Veneto was part of the Habsburg Empire until1866) in the middle of the last century.

All the most popular analytical methods of the time (e.g. the reactions ofTrommer, Moore, Fehling, Heller) were used, demonstrating that scientific andtechnical information travelled quickly around Europe. The scientific notespublished in medical journals reported about the analyses and about theprofessionals who supervised them. Medical doctors, directly or indirectlyconnected to the Giessen School of Liebig, and who had graduated in pharmacyand in chemistry, carried out the analyses as was the case at the same time inGermany and in Austria [14].

The Clinical Laboratory of Venice Hospital was founded in 1863 and its firstdirector was Giovanni Bizio, who was among the first group that graduated inchemistry at the University of Padua, and who was one of the fathers of clinicalchemistry in Northern Italy [12].


Acknowledgements

The authors acknowledge the helpful and valuable assistance of the colleaguesin the AACC History Division, Dr. Samuel Meites, Prof. Paul Wolf and Dr.Nathan Radin.

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Diagnosis of diabetes mellitus at the Hospital of Venice in 1863