Liver electrolysis: pH can reliably monitor the extent of hepatic ablation in pigs.

Electrolysis is a method of tissue ablation that creates chemical species and a pH gradient in response to direct current. Initial studies of electrolysis in animal models and humans have shown that it is a safe, predictable and effective process for destroying normal and tumour-bearing liver in a linear, dose-dependent manner. Presently, the amount of current that is applied (in coulombs) has to be calculated using historical data, with inherent inaccuracy. The present study tested whether pH could be used as a real-time monitor in order to predict more accurately the extent of necrosis. A total of 70 electrolytic lesions were created in 14 pigs, with pH monitoring of the lesion edge. The normal range of pH values was 6.5-8.7. A pH of less than 6 (at the anode) or more than 9 (at the cathode) reflected total cellular necrosis. When a pH value was recorded between 6.0 and 6.5 at the anode or between 8.7 and 9.0 at the cathode, the presence of necrosis was variable. In conclusion, during electrolytic ablation, pH measurement can monitor the extent of the induced necrosis.

Related papers:

title: A collective review of the world literature on hepatic cryotherapy.
abstract: Published results of hepatic cryotherapy are now available for almost 900 patients. Its safety is well established and its clinical role in treating patients with unresectable hepatoma or liver metastases from colorectal carcinoma is well supported by tumour marker and survival data; the results in the treatment of neuroendocrine liver metastases are promising. Its role as an alternative to liver resection is not yet well supported by long-term data. Although different adjuvant treatment protocols have been used following the cryotherapy of colorectal liver metastases, the effect of adjuvant treatment on recurrence or survival has not been assessed in prospective studies. Laparoscopic hepatic cryotherapy is feasible in selected patients with suitable tumour locations. However, the proportion of patients who might be usefully treated with this technique is not yet well established. The mechanisms of tissue destruction by freezing are reviewed.

title: Clinical score for predicting recurrence after hepatic resection for metastatic colorectal cancer: analysis of 1001 consecutive cases.
abstract: There is a need for clearly defined and widely applicable clinical criteria for the selection of patients who may benefit from hepatic resection for metastatic colorectal cancer. Such criteria would also be useful for stratification of patients in clinical trials for this disease.



Clinical, pathologic, and outcome data for 1001 consecutive patients undergoing liver resection for metastatic colorectal cancer between July 1985 and October 1998 were examined. These resections included 237 trisegmentectomies, 394 lobectomies, and 370 resections encompassing less than a lobe. The surgical mortality rate was 2.8%.



The 5-year survival rate was 37%, and the 10-year survival rate was 22%. Seven factors were found to be significant and independent predictors of poor long-term outcome by multivariate analysis: positive margin (p = 0.004), extrahepatic disease (p = 0.003), node-positive primary (p = 0.02), disease-free interval from primary to metastases <12 months (p = 0.03), number of hepatic tumors >1 (p = 0.0004), largest hepatic tumor >5 cm (p = 0.01), and carcinoembryonic antigen level >200 ng/ml (p = 0.01). When the last five of these criteria were used in a preoperative scoring system, assigning one point for each criterion, the total score was highly predictive of outcome (p < 0.0001). No patient with a score of 5 was a long-term survivor.



Resection of hepatic colorectal metastases may produce long-term survival and cure. Long-term outcome can be predicted from five criteria that are readily available for all patients considered for resection. Patients with up to two criteria can have a favorable outcome. Patients with three, four, or five criteria should be considered for experimental adjuvant trials. Studies of preoperative staging techniques or of adjuvant therapies should consider using such a score for stratification of patients.

title: Resection of colorectal liver metastases
abstract: From 1960 to 1992 a total of 1718 patients with liver metastases from colorectal carcinoma were recorded. Of these patients, 469 (27.3%) underwent hepatic resection, which was performed with curative intent in 434 patients (25.3%). Operative mortality in this group was 4.4%, being 1.8% (2 of 114) during the last 3 years. Significant morbidity was observed in 16% of patients with a decrease to 5% (6 of 112) for the last 3 years. A 99.8% follow-up until November 1, 1993 was achieved. Excluding operative mortality, there are 350 patients with “potentially curative” resection and 65 corresponding patients with minimal macroscopic (n=19) or microscopic (n=46) residual disease. The latter group demonstrated a poor prognosis, with median and maximum survival times of 14.4 and 56.0 months, respectively. Among the 350 patients having potentially curative resection, the actuarial 5-, 10-, and 20-year survivals were 39.3%, 23.6%, and 17.7%, respectively. Tumor-free survival was 33.6% at 5 years. In the univariate analysis, the following factors were associated with decreased crude survival: presence and extent of mesenteric lymph node involvement (p=0.0001); grade III/IV primary tumor (p=0.013); synchronous diagnosis of metastases (p=0.014); satellite metastases (p=0.00001); metastasis diameter of>5 cm (p=0.003); preoperative carcinoembryonic antigen (CEA) elevation (p=0.03); limited resection margins (p=0.009); extrahepatic disease (p=0.009); and nonanatomic procedures (p=0.008). With respect to disease-free survival, extrahepatic disease (p=0.09) failed to achieve statistical significance, whereas patients with primary tumors in the colon did significantly better than those with rectal cancer (p=0.04). The presence of five or more independent metastases adversely affected resectability (p<0.05). However, once a radical excision of all detectable disease was achieved, no significant predictive value of an increasing number of metastases (1–3 versus≥4) on either overall (p=0.40) or disease-free (p=0.64) survival was found. Using Cox's multivariate regression analysis, the presence of satellite metastases, primary tumor grade, the time of metastasis diagnosis diameter of the largest metastasis, anatomic versus nnanatomic approach, year of resection, and mesenteric lymph node involvement each independently affected both crude and tumor-free survival.RésuméEntre 1960 et 1992, un total de 1718 patients ayant des métastases d'un cancer d'origine colorectale ont été enregistrés. Quatre cent soixante-neuf patients (27.3%) ont eu une résection hépatique qui a été effectuée avec une intention curatrice chez 434 (25.3%_. La mortalité opératoire a été de 4.4%, mais seulement de 1.8% pendant ces trois dernières années (2/114). La morbidité globale a été de 16%, et de 5% pendant ces trois dernières années (5/112). A la date du 1 Novembre 1993, on avait des nouvelles de 99.8% des patients. Après exclusion des patients décédés pendant l'intervention, 350 patients ont eu une résection «potentiellement» curatrice. Parmi ceux-là, 65 étaient le siège de tumeur résiduelle soit macroscopique (n=19) soit microscopique (n=46). Parmi ces derniers, le pronostic était moins bon car la médiane et le maximum de survie a été respectivement de 14.4 et 56 mois. Parmi les 350 patients ayant une cure «potentiellement» curatrice, la survie actuarielle à 5, 10 et à 20 ans a été respectivement de 39.3%, de 23.6% et de 17.7%. La survie sans tumeur a été de 33.6% à 5 ans. En analyse univariable, les facteurs suivants ont été associés avec une diminution de survie globale: présence de métastase lymphatique mésentérique étendue (p=0.0001), tumeur primitive stade III/IV (p=0.013), métastase synchrone (p=0.014), métastases satellites (p=0.00001), diamètre de métastase>5 cm (p=0.003), taux d'ACE élevé (p=0.03), marges de résection limitée (p=0.009), cancer extrahépatique (p=0.0009), et intervention non-anatomique (p=0.008). En ce qui concerne la survie sans tumeur, la présence de cancer extra-hépatique n'a pas atteint de signification statistique, alors que les patients avec un cancer primitif du côlon avaient un pronostic meilleur que ceux qui avaient un cancer du rectum (p=0.04). La présence de cinq métastases indépendantes ou plus influençait la résequabilité en sens inverse (p<0.05). Une fois qu'une résection radicale de toute maladie détectable a été faite, le nombre de métastases (1–3 vs. 4 ou+) n'avait plus de valeur prédictive sur ni la survie globale (p=0.40) ni la maladie sans métastases (p=0.64). En utilisant le modèle de Cox, la présence de métastases satellites, le stade de la tumeur primitive, le moment du diagnostic de métastases, le plus grand diamètre, de métastase, l'approche anatomique ou pas, l'année de la résection et la présence d'envahissement lymphatique mésentérique ont tous été des facteurs indépendants influençant la survie globale et la survie sans tumeur.ResumenEn el período 1960 a 1992 registramos un total de 1.718 pacientes con metástasis hepáticas de carcinomas colorectales; 469 (27.3%) fueron sometidos a resección, la cual fue realizada con propósito curativo en 434 pacientes (25.3%). La mortalidad en este grupo fue 4.4%, siendo 1.8% (2 de 114) en los últimos 3 años. Se observó morbilidad significativa en 16% de los casos con una disminución a 5% (6 de 112) en los últimos 3 anños. Hasta noviembre 1 de 1993, se logró un 99.8% de seguimiento. Excluyendo la mortalidad operatoria, hay 350 pacientes con resección “potencialmente curativa” y 65 pacientes con enfermedad residual macroscópica mínima (n=19) o microscópica (n=46). Este último grupo demuestra el pronóstico tan pobre con una sobrevida media y máxima de 14.4 y 56 meses, respectivamente. Entre los 350 pacientes que tuvieron una resección potencialmente curativa la tasa actuarial de sobrevida a 5, 10 y 20 años fue 39.3%, 23.6% y 17.7%, respectivamente. La tasa de sobrevida libre de tumor fue de 33.6% a 5 años. En el análisis univariable, los siguientes factores aparecieron asociados con disminución de la tasa cruda de sobrevida: presencia o extensión de la invasión ganglionar mesentérica (p=0.0001), tumor primario grado III/IV (p=0.013), diagnóstico sincrónico de metástasis (p=0.014), metástasis satélites (p=0.00001), diámetro de las metástasis>5 cm (p=0.003), elevación preoperatoria del CEA (p=0.03), márgenes limitados de resección (p=0.009), enfermedad extrahepática (p=0.009) y procedimiento no anatómico (p=0.008). Con respecto a la sobrevida libre de enfermedad, las lesiones extrahepáticas (p=0.09) demostraron no tener significancia estadística; los pacientes con tumores primarios del colon evolucionaron significativamente mejor que los pacientes con cáncer rectal (p=0.04). La presencia de 5 o más metástasis independientes afecta la resecabilidad (p<0.05). Sin embargo, una vez lograda la resección radical de todas las metástasis, no se encontró un valor significativo de predicción según un númeo creciente de metástasis (1–3 vs ≥ 4) sobre la tasa global de sobrevida (p=0.40) o sobre la tasa de sobrevida libre de enfermedad (p=0.64). En el análisis multivariable de regresión de Cox se encontró que los siguientes factores afectan en forma independiente la tasa cruda de sobrevida y la tasa de sobrevida libre de tumor: presencia de metástasis satélites, grado del tumor primario, momento del diagnóstico de las metástasis, diámetro de la mayor de las metástasis, abordaje anatómico vs no anatómico, año en que se efectuó la resección e invasión de los ganglios linfáticos mesentéricos.

title: Treatment of colorectal liver metastases
abstract: Surgical resection is the only potentially curative treatment for colorectal liver metastases, with 5‐year survival rates approaching 40 per cent. However, at present only 20–25 per cent of such lesions are deemed resectable. This review examines developments in neoadjuvant and adjuvant treatments of colorectal liver metastases that aim to improve the results of surgical management of this disease.

title: Liver resection and laser hyperthermia.
abstract: Table 2 provides a comparison of these interstitial and in situ techniques, assessing several criteria and utilizing and expanding on an excellent review by Masters et al. The rapid advance of technology and cross-fertilization between groups using different interstitial techniques will lead to a clear understanding of the benefits and limits of each. However, there is essentially no information at the present time to suggest that these techniques should be used in lieu of hepatic resection in an attempt to cure patients who are good operative risks. There are insufficient data of a controlled nature to determine that there has been a survival or palliative benefit in many of the patients so treated. Nevertheless, as it is clear that these treatments cause tissue destruction in an appropriate nonmorbid way and that they are well tolerated with low risk to the patients, it is entirely conceivable that interstitial techniques will replace hepatic resection in some instances in the future, particularly for lesions smaller than 3 cm.

title: Role of interstitial therapy in the treatment of liver cancer
abstract: Conventional palliative management of inoperable focal hepatic tumours remains unsatisfactory. Interstitial techniques such as cryotherapy, alcohol injection, low power laser hyperthermia and interstitial radiotherapy offer alternative approaches. Cryotherapy is an effective and precise technique for inducing tumour necrosis. It can only be performed at laparotomy making it relatively invasive and retreatment impractical. Alcohol is cheap and can be injected percutaneously. However, inhomogeneous distribution produces imprecise and non‐reproducible lesions. Low power laser hyperthermia produces precise and reproducible areas of necrosis that are roughly spherical in shape. At present, this technique is most effective for small tumours. Interstitial radiotherapy remains the least evaluated of all the interstitial techniques. Unlike cryotherapy and low power laser hyperthermia, the biological effect of ethanol injection and interstitial radiotherapy cannot be monitored in real time by ultrasound. With the exception of cryotherapy, all methods can be applied percutaneously with low morbidity and mortality. None of these techniques is established, but they may offer the prospect of cure in cases where all areas of tumour can be positively identified and fully treated. However, in most instances the intention is to control the growth of relatively small discrete volumes of tumour within the hepatic parenchyma.

title: Effect of low level direct current on in vivo tumor growth in hamsters.
abstract: A preliminary study has been carried out on the effect of low level direct current on tumor growth using an experimental tumor model developed from an amelanotic melanoma (T1-4) in the hamster. An inoculum of 2 X 10(6) viable cells was injected s.c. on day 0; on day 7 the tumor-bearing animals were randomly divided into treatment and control groups. On days 7 through 11 inclusive, the treatment group was subjected to electrical current (direct current) at levels from 0.1 to 2.4 mA, for 1 h/day under general anesthesia. Control groups were subjected to the same procedures, with the exception that the electrodes were not connected to the current source. On day 14, the animals were killed and autopsied; their tumors were removed, weighed, and sectioned. Treated tumors decreased in mass (as a percentage of controls) from 89% at 0.1 mA to 2% at 2.4 mA. Increased necrosis of the treated tumors was noted macroscopically and microscopically. On histological examination, it was observed that a thin rim of viable cells remained around the periphery after treatment even at the highest current levels. Similar results were obtained with both stainless steel and platinum-30% iridium electrodes. In separate experiments where the animals were allowed to survive after a treatment period (1 h/day for 5 days at 2.4 mA), the viable cells at the periphery developed into tumors whose mass at 28 days posttreatment averaged only 52% of that of the control tumors. The mechanism of growth reduction is unknown but hyperthermia was shown not to be a factor.

title: Electrochemically induced hepatic necrosis: the next step forward in patients with unresectable liver tumours?
abstract: The treatment of patients with unresectable liver tumours remains an unsolved clinical problem. Several methods of locoregional treatment have been developed. These methods rely mainly on direct thermal or chemical insults and consequently have their own inherent limitations in clinical usage. The 'ideal' treatment would combine the direct cytotoxic effects of chemical treatments with the relative predictability of thermal insults, without the associated complications. This study aims to investigate whether the direct chemical effect of electrolytic hepatic necrosis is associated with any heating effect, and if so, whether the temperature change is dose-dependent.



An electrolytic 'dose' sufficient to induce a localized zone of hepatic necrosis was delivered to the livers of rats and pigs via implanted platinum electrodes.



The results showed that there was no significant temperature increase at low current levels (2-4 mA) in the rat liver. In the pig, there was a significant (P < 0.01) increase in temperature of 4.2 degrees C during electrolysis, when delivered at between 20 and 50 mA. However, such a small increase in temperature would have been insufficient to cause appreciable thermal necrosis.



This study demonstrates that electrolysis-induced hepatic necrosis is produced without an increase in temperature; clearly cell death results from the direct effects of cytotoxic electrode products and an alteration of intracellular pH. Consequently, it is likely that as a method for ablating liver tumours, electrolysis should be associated with fewer complications than other forms of locoregional treatment.

title: Tumor treatment by direct electric current-tumor temperature and pH, electrode material and configuration
abstract: Electrotherapy with low-level direct current has been suggested as an effective regional cancer treatment. A great variety of electrode materials, their placement with respect to tumor (i.e. electrode configurations) current levels and therapeutic schedules have been employed to date. In our paper the impact of electrode materials and configurations was studied, employing the same treatment parameters and schedule, on the subcutaneously grown solid tumor fibrosarcoma SA-1 in A/J mice. The effect of electrotherapy was assessed by tumor volume determination on each consecutive day and by determination of the extent of necrosis along the tumor's greatest diameter immediately after electrotherapy, 24, 48 and 72 hours later. The development of tumor necrosis after electrotherapy application differed for different electrode configurations. Intratumoral temperature and pH were measured before and after the application of electrotherapy in all electrode configurations used. Irrespective of the electrode configuration the intratumoral temperature was unchanged by electrotherapy. Considerable variations in pH were revealed for configurations where one electrode was inserted into the tumor, whereas in configurations with both electrodes outside, the tumor pH remained unchanged by electrotherapy. Electrodes made of platinum, platinum (90%) :iridium (10%) alloy, gold, silver and titanium were used for current delivery, and significant tumor growth retardation was achieved irrespective of electrode configuration and material (p< 0.001, Mann-Whitney Rank-Sum test). In addition to possible toxicity arising from dissolved metal ions and products of electrochemical reactions, which may result from electrolysis, direct effects of current on tumor and immune cells are suggested.

title: Low-level direct electrical current therapy for hepatic metastases. I. Preclinical studies on normal liver.
abstract: Low-level direct electrical current has shown promise as a potential therapeutic modality (direct current therapy; DCT) in the treatment of malignant disease, including metastases, but to date much experimental work has been empirical and has added little to our knowledge of the mechanisms involved. As a prerequisite to a clinical trial for metastases in the liver, we have employed an in vivo liver model to examine the quantitative and qualitative relationships between electrode polarity, charge and tissue necrosis. Two distinct regions of necrosis were induced, distinguishable histologically and by magnetic resonance imaging: (i) a cylindrical region of primary necrosis centred on the electrode, its volume directly proportional to the charge passed, but greater at the anode than cathode; and (ii) a wedge-shaped infarct, apex at the electrode and base extending to the liver edge. The extent of this infarct was again greater at the anode than the cathode, but showed a sigmoidal relationship with charge. Results indicate pH changes at the electrodes as likely mediators of tissue injury, but show also that significant distant ischaemic injury can occur as a consequence of primary damage. These findings should be considered when selecting tumours for possible direct current therapy and when determining the sites of electrode placement.

title: A new treatment for unresectable liver tumours: long-term studies of electrolytic lesions in the pig liver.
abstract: The majority of liver tumours are inoperable and an alternative treatment to surgical resection is urgently needed. Electrolysis has been investigated in a rat model and the procedure is safe, with accurate and predictable effects. The necrosis produced has also been shown to cause destruction of tumour deposits in the rat liver. A similar evaluation in a large animal model was necessary before clinical trials could commence. Using platinum electrodes connected to a d.c. generator, areas of hepatic necrosis were created in the pig liver. Animals were killed at various time points after treatment to assess the extent of healing. Treatment was uneventful and all animals made a full recovery. No animal died from the treatment or had to be killed prematurely. After 2 days of treatment, healing was minimal but at successive time points there was progressive evidence of healing, such that after 4 months, the original electrolytic lesion was greatly reduced in size and the large area of necrosis seen at the early time points was largely replaced by a fibrous scar with only small islands of necrotic tissue. In a large animal model, electrolysis is a safe method for creating areas of hepatic necrosis. The lesions heal with time and are associated with minimal morbidity. The results support a trial of electrolysis in patients with unresectable liver tumours.

title: Experimental study of electrolysis‐induced hepatic necrosis
abstract: One of the most promising but unexplored methods for treating patients with irresectable liver tumours is electrolysis. This study examined the effect of increasing ‘current dose’ on the volume of the lesion induced in normal rat liver.

title: Electrolytic Ablation of Colorectal Liver Metastases: 1-Year Histological Patient Follow-Up
abstract: Whilst up to 50% of patients with colorectal carcinoma will develop liver metastases, only 20% of those patients will be amenable to potentially curative liver resection. There is therefore great need for other effective therapies, and we report complete ablation of a colorectal liver metastasis using electrolysis, which was histologically confirmed at 1 year. The clinical impact of complete tumour ablation at 1 year means that a proportion of those patients currently deemed inoperable and therefore incurable, may be suitable for curative treatment.

title: The effects of low-level direct current therapy on a preclinical mammary carcinoma: tumour regression and systemic biochemical sequelae.
abstract: Low-level direct electric current has been shown to be capable of destroying tumour tissue. Using an early-passage subcutaneous murine mammary carcinoma, the relationships between the volume of tumour destruction, charge and polarity have been examined. The results revealed a direct correlation between charge passed and absolute volume regression when the intratumoral electrode was made either an anode or a cathode. Tumour destruction for a given charge was significantly greater following anodic than cathodic treatment. A direct correlation was also observed between the percentage volume of prompt treatment-induced regression and the in situ end point of tumour growth delay. During the course of these experiments, a highly reproducible toxic effect was discovered, which has not been previously reported for this modality. An anodic charge greater than 10.6 coulombs or a cathodic charge greater than 21.6 coulombs resulted in 100% mortality at 24-72 h, while lower charges had no influence on mortality. Quantitative assays of a number of blood parameters showed that mortality was associated with serum electrolyte imbalances and appeared to be the result of the metabolic load of tumour breakdown products. These effects are similar to the tumour lysis or surgical crush syndromes and should not constitute a significant problem in clinical practice, where the tumour mass to total body mass ratio will normally be much smaller.

title: Electrochemical aspects of treatment of tissue with direct current.
abstract: In the electrochemical treatment of tissue with direct current the main reactions at the anode are the formation of oxygen in addition to acidification due to liberated hydrogen ions, and formation of chloride, if platinum is used as anode material. At the cathode hydrogen is formed and hydroxide ions are liberated. Calculations have been made for the two major anodic reactions. As a first approximation just one-dimensional diffusion is considered corresponding to the use of plate electrodes. It appears from these calculations that the liberated hydrogen ions determine the extension of the locally destroyed zone around the anode. The destructive effect of chlorine probably occurs in an inner zone close to the anode.

title: The laryngeal mask airway in experimental pig anaesthesia
abstract: The pig is used as a large animal model in many research projects. Standard practice for airway maintenance under general anaesthesia is using endotracheal (ET) intubation after intravenous induction to a near surgical plane. This is a technically demanding skill, requiring the assistance of an experienced technician. A technique is required which simplifies pig anaesthesia. This study examined the feasibility and potential advantages of using the laryngeal mask airway (LMA) in 10 pigs during laparotomy under spontaneous breathing anaesthesia. The results show that the LMA can be inserted rapidly, with minimal time for airway control by researchers relatively inexperienced in anaesthesia and is associated with few complications. By removing the need for intravenous induction, an entire step in the anaesthetic process is removed. The LMA designed for humans fits well in the pig hypopharynx; all pigs could be manually ventilated with no detectable gas leak. Although the pigs in this study were spontaneously breathing it is proposed that the LMA should be further investigated in studies of artificially ventilated pigs.

title: Electrolytic Treatment of Colorectal Liver Tumour Deposits in a Rat Model: A Technique with Potential for Patients with Unresectable Liver Tumours
abstract: Patients with unresectable malignant liver tumours have a poor prognosis. A technique is needed which improves long-term survival. Previous studies in the rat have shown that electrolysis is a safe, predictable and reproducible method for creating areas of necrosis in the normal rat liver. This study examined the effects of electrolysis on colorectal liver ‘metastases’ in the rat.  Tumours of colorectal origin were implanted into the livers of Wistar-WAG rats. Two weeks after implantation the tumours were treated with electrolysis. A direct current generator, connected to 2 platinum intrahepatic electrodes was used to examine the effects of various electrode configurations on the extent of tumour necrosis.  Significant (p<0.001) tumour ablation was achieved with all electrode configurations. Tumour necrosis was more complete (p<0.05) with the electrodes positioned on either side of the tumour than with both electrodes placed in the centre of the tumour. Liver enzymes (AST and ALT) were significantly (p<0.001) elevated after treatment, but returned towards normal by 2 days.  This study has shown that colorectal liver ‘metastasis’ can be ablated by electrolysis in a rat model. Two separate mechanisms of tumour ablation were observed: With the electrodes directly in or adjacent to the tumour, necrosis resulted from the action of cytotoxic electrode products, whereas by positioning the electrodes proximal to the tumour, necrosis was induced by a ‘secondary’ ischaemic effect. The findings confirm the view that electrolysis has great potential for treating patients with unresectable malignant liver tumours.

title: The paradigm of biologically closed electric circuits (BCEC) and the formation of an International Association (IABC) for BCEC systems.
abstract: Matter is condensed energy. Matter derived from electromagnetic energy, supplied with the electrically powered BCEC systems (biologically closed electric circuits) inherits prerequisites to become biological matter. This is possible because mechanisms of BCEC systems contain the capacity to initiate structuring and functioning of matter. The principle of BCEC systems, their actual and potential importance for structure and function in biology and medicine, has been presented only in the form of a survey. Only few examples of the impact of the BCEC systems have been described. For detailed information, the reader is advised to take part of the original background articles by means of the reference list, which extends beyond the selected descriptions. Thereby it is hoped that the reader may get an understanding of the central biological role of the BCEC systems. They represent in this partial theory of the biological evolution a key mechanism which may provide the important primary steps that are necessary for the transfer of non-biological into biological matter. Second, also other factors are evidently contributing to biological differentiation, including for instance the principle of differential selection of species. Due to their basic role, the BCEC systems can nevertheless be recognized to be involved in the majority of structural and functional expressions in biology. This rests on the fact that our physical world once developed from energy and specifically its representation of electric energy. It has also been emphasised that electric energy is equivalent to the remarkable Oriental concept of Qi ("life energy"). Differences are predominantly a matter of semantics.

title: Factors influencing survival of patients undergoing hepatectomy for colorectal metastases
abstract: The extent of surgical margin is still a matter of controversy with regard to outcome after liver resection for metastatic disease. The aim of this study was to clarify the significant prognostic factors after hepatectomy for colorectal metastases, with special reference to the surgical margin.

title: Biologically Closed Electric Circuits: Activation of Vascular Interstitial Closed Electric Circuits for Treatment of Inoperable Cancers
abstract: Normal metabolic potential differences between tissue regions supply energy for chemical transports. Some of these transports take place via a circulatory system that is coupled to the non-selective bulk transports of blood and lymph circulation. It is called the vascular-interstitial closed circuit (VICC) and represents a key mechanism for healing of injured tissue. By applying direct current between electrodes implanted into tissues it is possible to enhance the VICC transports and to induce healing in inoperable cancers in the lungs, e.g., multiple metastases. To date 26 pulmonary cancers in 20 patients have been treated, 12 of which have regressed, without subsequent regrowth (2 to 5 year follow-up period). the technique of treatment is relatively simple and only slightly distressing to the patients. No fatalities have occurred.

title: Electroporation Therapy of Head and Neck Cancer
abstract: The purpose of this paper is to introduce the concept of electroporation therapy and present our results from using this new technique combined with intralesional bleomycin in head and neck cancer patients. Electroporation therapy is a technique wherein high-voltage electric impulses delivered into a neoplasm transiently increase cell membrane permeability to large molecules, including cytotoxic agents. In this phase I/II study, extremely low-dose bleomycin sulfate was electroporated into head and neck malignant neoplasms in 10 patients. Tumor responses included 2 nonresponders, 3 partial responders, and 5 complete responders, with a mean follow-up of 40 weeks. We conclude that this technique offers promising possibilities in the local treatment of head and neck cancer.

title: Electrochemical modification of tumor growth in mice.
abstract: We evaluated the effect of direct electrical current on large tumors in mice. Lewis lung carcinoma greater than 1 cm in the shortest dimension was treated percutaneously with 20 mA for 15 min. Separate groups were given one or more than one (two or three) percutaneous electrical treatments (PET). A third group was given sham electrical treatment, and a fourth group had surgical excision of the tumor. Animals in both PET groups survived longer and had smaller primary tumors at death compared with the sham group. PET did not alter the systemic course of the disease, judged by lung and spleen weights and by histological observation of the extent of metastatic burden in the lung. Surgery resulted in long-term survival of 17% and an increase in average survival time compared with both PET and sham treatment. PET produced rapid and polarity-dependent alterations in physiological solutions in vitro, and it is likely that similar electrochemical processes mediated the observed reduction in tumor growth. PET is potentially useful as an adjuvant modality because it reduces local tumor mass but does not alter the extent of metastasis.

title: Cytotoxicity of weak electrolytes after the adaptation of cells to low pH: role of the transmembrane pH gradient.
abstract: Theory suggests that the transmembrane pH gradient may be a major determinant of the distribution of lipophilic weak electrolytes across the cell membrane. The present study evaluates the extent to which this factor contributes to pH-dependent changes in the cytotoxicity of two such chemotherapeutic drugs: chlorambucil and mitoxantrone. Experiments were performed with two cell types of the same origin but exhibiting different pH gradients at the same extracellular pH (pHe): CHO cells cultured under normal physiological conditions (pH 7.4) and acid-adapted cells obtained by culturing under low pH conditions (6.8). Over the pHe range examined (6.0-7.6), the difference between intracellular pH (pHi) and pHe increased with decreasing pHe. Acid-adapted cells were more resistant to acute changes in pHi than normal cells, resulting in substantially larger gradients in these cells. Drug cell survival curves were performed at pHe values of 6.4, 6.8 and 7.4. The cytotoxicity of chlorambucil, a weak acid, increased with decreasing pHe, and low pH-adapted cells were more sensitive than normal cells at the same pHe. In contrast, for the weak base, mitoxantrone, cytotoxicity increased with pHe and was more pronounced in normal cells. As predicted by the theory, the cytotoxicity of both drugs changed exponentially as a function of the pH gradient, regardless of cell type. For mitoxantrone, the rate of such change in cytotoxicity with the gradient was approximately two times greater than for chlorambucil. This difference is probably due to the presence of two equally ionizable crucial groups on mitoxantrone vs one group on chlorambucil. It is concluded that the cellular pH gradient plays a major role in the pH-dependent modulation of cytotoxicity in these weak electrolytes. The data obtained also suggest that a pronounced differential cytotoxicity may be expected in vivo in tumour vs normal tissue. In comparison with normal cells at a pHe of 7.4 (a model of cells in normal tissues), acid-adapted cells at a pHe of 6.8 (a model of cells distal from supplying blood vessels in tumours) were more sensitive to chlorambucil, with a dose-modifying factor of approximately 6, and were more resistant to mitoxantrone by a factor of 14.

title: On the use of monocrystalline antimony pH electrodes in gastro-oesophageal functional disorders
abstract: Monocrystalline antimony electrodes have been shown to be suitable for thein vivo determination of pH in blood, tissue and in the upper gastro-intestinal canal. Thanks to their small dimensions it has been possible to mount them into conventional manometry catheters for oesophageal investigation. The monocrystalline antimony pH electrode has several advantages over the conventional pH glass electrode; better accuracy, shorter rise time, smaller dimensions. The monocrystalline antimony electrode has been used for long-term registration of gastro-oesophageal reflux, for the oesophageal acid clearing test and for identification of the pH gradient zone between the gastric and oesophageal mucosa. Its use in combination with pressure sensors has added a new dimension to the diagnosis of functional disorders in the gastro-oesophageal region.

title: Improved antimony-antimony (III) oxide pH electrodes
abstract: In routine clinical care rapid results from pH determinations are often desired. This calls for continuousin vivo measurements. Today only intermittentin vitro pH analysis is routinely performed, as the existing transducers do not meet the clinical demands on dimensions, stability, stabilisation time, resolution and ruggedness. Here, some results from a basic experimental study on antimony-antimonyoxide pH electrodes are presented. The purpose of the investigation has been to gain some information on the electrode processes, which govern the electrode response, and to use this information to synthesise more stable and reproducible electrodes than those presently existing. These electrodes are intended, in an appropriate miniaturised form, forin vivo routine pH determinations on homo, e.g in a continuous acid-base balance surveillance application.SommaireDans les traitements cliniques routiniers, on désire souvent déterminer rapidement le pH. Pour cela, il faut effectuer des mesures continuesin vivo. Aujourd'hui, seule une analyse de pH intermittentein vitro est effectuée de manière routinière, étant donné que les transducteurs existants ne peuvent résolution et la robustesse. Cet article présente certains résultats d'une étude expérimentale fondamentale sur des électrodes de pH du type Sb/monoxyde de Sb. Le but de l'étude était d'obtenir certains renseignements sur les processus auxquels sont soumises les électrodes et qui gouvernent leur réponse, pour pouvoir utiliser ces renseignements en vue de la synthèse d'électrodes plus stables et donnant des résultats plus reproductibles que celles disponibles actuellement. Ces électrodes sont destinées sous une forme suffisamment miniaturisée à déterminer le pHin vivo sur l'homme de manière routinière, c'est-à-dire dans des applications de surveillance continue de l'équilibre acides/ bases.ZusammenfassungBei der klinischen Routinebehandlung sind pH-Bestimmungen oft wünschenswert. Hierzu werden andauerndein vivo Messungen benötigt. Heutzutage werden nur unterbrochenein vitro Analysen als Routineuntersuchungen durchgeführt, da die vorrätigen Meßwandler nicht den klinischen Anforderungen mit Hinsicht auf Größe, Stabilität, Stabilisationszeit, Resolution und Robustheit nachkommen. Hier werden einige Resultate aus einer experimentellen, Grundstudie an Antimon-Antimonoxid-pH-Elektroden dargestellt.

title: New approach for elucidating the oxygen sensitivity and calibration of the antimony electrode
abstract: The biomedical use of the polycrystalline antimony pH electrode is hampered by oxygen dependence and low pH resolution (0.1 pH). Although, the pH resolution is improved by using highly purified crystallographically oriented monocrystalline antimony (COMA), the oxygen effect on the electrode remains. In an in vitro examination of the COMA electrode it was found that the oxygen dependence is similar to that of polycrystalline antimony, i.e. nonlinear with an increase in sensitivity below 10 kPa. These findings imply that the oxygen tension in the vicinity of a COMA pH electrode, has to be known or controllable for the pH reading to be at all valid especially at low oxygen levels, such as is often seen in biomedical applications. A calibration procedure using a zero oxygen solution is used in order to obtain absolute pH readings in a measurement solution of pre-set or known oxygen levels. Furthermore, the nonlinear oxygen sensitivity of the antimony electrode suggests that the underlying electrochemical reactions are other than those described by corrosion theory.

title: Measurement of tissue pH for predicting viability in pedicle flaps: Experimental studies in pigs
abstract: A new method for predicting viability of single pedicle skin flaps is reported. Measuring hydrogen ion concentration within the tissue reveals the degree of anaerobic metabolism in cases of inadequate circulation. Experimental studies in the pig showed a significant pH difference between the tissue which became necrotic and the tissue which survived. The measurement of pH can be applied to human tissues but criteria for tissue survival in man have yet to be established.

title: MONITORING RESPONSE TO CHEMOTHERAPY OF INTACT HUMAN TUMOURS BY 31P NUCLEAR MAGNETIC RESONANCE
abstract: 10 kidneys bearing adenocarcinoma (hypernephroma), 1 ureteric tumour, 2 Wilms' tumour from patients undergoing nephrectomy, and normal human and animal kidneys were preserved and later tested to see whether normothermic blood perfusion restored glomerular filtration, oxygen consumption, and energy metabolism, as monitored by 31P nuclear magnetic resonance (NMR). NMR spectra of normal human kidney and of renal tumours were characteristic of kidney in other species (rat, rabbit, and dog), with six principal resonances assigned to adenosine monophosphate, inorganic phosphate (Pi), glycerophosphoryl choline, gamma, alpha, and beta adenosine triphosphate and an intrarenal pH 7.0-7.2. 7 out of 9 hypernephromas and 1 of the 2 Wilms' tumours produced an additional resonance at +4.2 ppm. This peak was absent from all normal kidney spectra and may represent Pi in a specific and highly acidic environment (pH 6.1-6.5) found in actively metabolising human tumours. Furthermore, in 1 tumour (out of 5 tested) the +4.2 ppm peak rose rapidly after the addition of chemotherapeutic agents to the perfusing blood. This observation may indicate drug sensitivity of this one tumour. The results suggest that 31P NMR imaging may be used to monitor therapeutic response during regional perfusion of malignant tumours in man.

title: Blood flow, oxygen and nutrient supply, and metabolic microenvironment of human tumors: a review.
abstract: The objective of this review article is to summarize current knowledge of blood flow and perfusion-related parameters, which usually go hand in hand and in turn define the cellular metabolic microenvironment of human malignancies. A compilation of available data from the literature on blood flow, oxygen and nutrient supply, and tissue oxygen and pH distribution in human tumors is presented. Whenever possible, data obtained for human tumors are compared with the respective parameters in normal tissues, isotransplanted or spontaneous rodent tumors, and xenografted human tumors. Although data on human tumors in situ are scarce and there may be significant errors associated with the techniques used for measurements, experimental evidence is provided for the existence of a compromised and anisotropic blood supply to many tumors. As a result, O2-depleted areas develop in human malignancies which coincide with nutrient and energy deprivation and with a hostile metabolic microenvironment (e.g., existence of severe tissue acidosis). Significant variations in these relevant parameters must be expected between different locations within the same tumor, at the same location at different times, and between individual tumors of the same grading and staging. Furthermore, this synopsis will attempt to identify relevant pathophysiological parameters and other related areas future research of which might be most beneficial for designing individually tailored treatment protocols with the goal of predicting the acute and/or long-term response of tumors to therapy.

title: Extracellular acidosis delays onset of cell death in ATP-depleted hepatocytes.
abstract: A fluorometric assay using propidium iodide total fluorescence was utilized to quantitate cell viability in hepatocyte suspensions continuously. For viable hepatocytes exposed to KCN, fluorescence was linearly proportional to lactate dehydrogenase release and to nuclear labeling by propidium iodide. In KCN-treated hepatocytes, iodoacetate eliminated the protective effect of the fed state and fructose against the onset of cell death. A model of cell death with KCN and iodoacetate was developed to mimic the ATP depletion of anoxia. This "chemical hypoxia" was used to investigate the role of pH in cell death. At pH 7.4, cell viability decreased to 10% after 120 min, whereas at pH 5.5-7.0, cell viability was 65-85%, nearly the same as normoxic cells. During chemical hypoxia under acidotic conditions, a return of pH to 7.4 resulted in a rapid acceleration of cell killing, a "pH paradox." Inhibition of Na+-H+ exchange increased survival, whereas promoting exchange of intracellular Cl- for extracellular HCO3- potentiated cell killing. Monensin, a Na+-H+ ionophore, potentiated cell killing at pH 7.4 but not at pH 6.2. The results show that extracellular acidosis markedly protects against cell killing after ATP depletion, an effect that appears mediated through cytoplasmic acidification.

title: Effects of metabolic acidosis on viability of cells exposed to anoxia.
abstract: The effects of metabolic acidosis were examined in isolated rat hepatocytes under substrate-free oxygenated or anoxic conditions. Lowering extracellular pH to 6.6 under aerobic conditions had no deleterious effects on the cells as determined by trypan blue exclusion, lactate dehydrogenase (LDH) release, cellular K+ and Ca2+ content, and ability to increase ATP levels after nutrients and adenosine were added to media. Cytosolic pH was measured in aerobic cells at varying extracellular pH using 6-carboxyfluorescein. By using values for cytosolic pH obtained in this manner together with 5,5-dimethyl[2-14C]oxazolidine-2,4-dione (DMO) distribution data, a method was derived for determining intramitochondrial pH. The pH gradient across the mitochondrial membrane was found not to change with a decrease in extracellular pH from 7.4 to 6.9. At pH 6.9 hepatocytes were protected against anoxic injury as compared with cells incubated at pH 7.5 or 6.6. This protection was manifested by a decrease in vital dye uptake and LDH release, maintenance of higher cellular K+ content, less stimulation of respiration with succinate, improved recovery of ATP levels after return to an oxygenated nutrient environment, and maintenance of normal cellular Ca2+ content after reoxygenation. Recovery of cellular ATP content was independent of ATP levels, total adenine nucleotide pool, and energy charge ratio at the end of the anoxic period. Measurement of cytoplasmic pH in anaerobic cells by [14C]DMO distribution showed progressive cellular acidification with lowering of extracellular pH. The protective effects observed at pH 6.9 are not unique to hepatocytes since isolated renal cortical tubules exposed to anoxia have improved ATP levels on reoxygenation at this pH when compared with tubules incubated at pH 7.5.