[advances in food research] volume 24 || histamine (?) toxicity from fish products

4DVANCtS IN F W U H F \ I . A R L H . VOL . 24

HISTAMINE (?) TOXICITY FROM FISH PRODUCTS

SALLY HUDSON ARNOLD AND W . DUANE BROWN

I . Nature of the Problcm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B . Symptomology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . Cases of Histamine Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D . Earlier Reviews . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

11 . Mechanisms of Fornlation of Histamine in Fish . . . . . . . . . . . . . . . . . . . . . . . . A . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B . Histamine Fonnation by “Autolyric” Enzymes . . . . . . . . . . . . . . . . . . . . . C . Detection of Bacterial Histidine Dccarboxylases . . . . . . . . . . . . . . . . . . . .

E . Occurrence of Histamine Formers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F . Free Histidine as a Histamine Prccursor . . . . . . . . . . . . . . . . . . . . . . . . . . . G . Histidine Decarboxylase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H . Bacterial Destruction of Histaniinc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 . “Saurinc” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . J . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Ill . Detection and Determination of Levels of Histamine in Fish . . . . . . . . . . . . . A . Guinea Pig Ileum Contraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

D . Bacteria Responsible for Histanline Formation . . . . . . . . . . . . . . . . . . . . . .

B . Other Bioassays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . Fluoromrtric Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

E . Colorimetric Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F . Enzymatic Isotopic Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

D . Gas-Liquid Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

G . Thin-Layer Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV . Relationship of Spoilage to Histarnine Forniation . . . . . . . . . . . . . . . . . . . . . . V . Unresolved Probleiiis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

A . Is Scombroid Toxicity due to Histamine? B . Improved Analytical Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . The Anserine and Carnosine Question D . Possiblc Synergists or Potentiators

. . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

E . Allowable Levels of Histamine in Fish . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

i14 i14 114 1 IS 120 121 121 121 122 122 124 124 125 128 129 130 130 130 131 131 133 133 134 134 135 139 139 141 141 142 146 147

I13 Copyright i@ 1978 hy Academic Press . Inr

All riphrs of repnduciion in any form reserved . ISBN 0-12-016424-8

Institute of M d n i . Rrsourw Depurtmritt of Food Scieitcx* und Trchnology

Uniwsity of CnliJt~rnin. Duvis. Cul$miu

I14 SALLY HUDSON ARNOLD AND W . DUANE BROWN

I . NATURE OF THE PROBLEM

A. INTRODUCTION

Histamine toxicity from fish products, often called “scombroid poisoning,” generally involves the ingestion of scombroid fish from the families Scom- beresocidae and Scombridae. [Note: Because of inconsistencies in the literature with respect to the scientific nomenclature of genera and species of scombroid fishes, we are following the guidelines suggested by Klawe (1976). In some cases, this has necessitated our changing the description given in a cited paper to that used by Klawe.] Scombroid fish include saury, tuna, bonito, seerfish, but- terfly kingfish, and mackerel. These fish normally contain large amounts of free histidine in their muscle tissue. The free histidine can, under certain conditions, be decarboxylated by some bacteria to produce high levels of histamine. Such levels of histamine may be reached before the fish appears spoiled or is organoleptically unacceptable. The consumption of both “fresh” and processed fish having significant levels of histamine has resulted in histamine toxicity and clinical illness.

B. SYMPTOMOLOGY

Early reports suggested that histamine poisoning was caused by food allergy or idiosyncrasy (Kawabata ef al., I955a). These theories were readily disproven because people were not affected by wholesome products of the same type. Furthermore, several histamine outbreaks occurred in which all of the people eating the food subsequently showed symptoms of histamine toxicity (note references in Section 1,C). A relationship between histamine levels and fish spoilage was recognized by Geiger (1953, who observed that the skin irritation experienced after handling spoiled fish was similar to the skin irritation produced by handling pure histamine.

Scombroid fish poisoning clinically resembles that of histamine poisoning intoxication, although controversy still exists as to whether histamine ingested orally is actually toxic (Halstead, 1967; Granerus, 1968; Douglas, 1970). His- tamine is believed to be detoxified by bacterial enzymes during its passage through the intestinal wall (Aiso e? al., 1958a). Some workers (Geiger, 1955; Ienistea, 1973) have suggested the possibility that ingestion of large amounts of histamine may overcome the intestinal barrier, with histamine thereby gaining access to the blood; see Section V,A for a more detailed discussion.

Whether histamine is the sole toxic factor or not, it is generally found at high concentrations in foods causing scombroid poisoning. Levels of histamine in excess of 100 mg% (100 mg free base per 100 gm of fish flesh) have often been associated with clinical illness (Legroux et al., 1946; Van Veen and Latuasan,

HISTAMINE (?) TOXICITY FROM FISH PRODUCTS I15

1950; Kawabata et a / . , 195%). Furthermore, 100 mg% is often considered the critical concentration for histamine poisoning (Simidu and Hibiki, 1955b; Ferencik, 1970; Anon., 1973d).

The onset of symptoms usually occurs several minutes to three hours after ingestion of toxic food (Sapin-Jaloustre, 1957). Typical incubation periods are less than one hour, although a wide variation can occur from individual to individual. For example, victims of an outbreak in Japan experienced incubation periods ranging from five minutes to five hours after consuming seasoned mackerel (Scomber juponicus) (Kawabata et ( I / . , 1955a).

The symptoms of histamine toxicity from fish products are characteristic and vary little from outbreak to outbreak. Some victims complain that the toxic food had a characteristic sharp or peppery taste (Halstead, 1967). The most consis- tently noted symptom is a flushing of the facial and neck area, causing a feeling of intense heat and general discomfort (Sapin-Jaloustre, 1957). The facial flush is principally caused by the dilating action of histamine on the small blood vessels, capillaries, and venules. The flush is followed by an intense, throbbing headache which becomes a continuous dull ache deep in the head, often centered in the frontal and temporal regions (Douglas, 1975). Cardiac palpitation occurs in many instances since the heart beats forcefully but ineffectively (Douglas, 1975). Dizziness, faintness, itching, burning of the mouth and throat, rapid and weak pulse, and inability to swallow are also common characteristics (Anon., 1975~) . Many victims develop a rash on the face and neck, accompanied by severe itching (Halstead, 1967; Anon., 1973~) .

Secondary symptoms, experienced by less than 25% of the victims, are gas- trointestinal in nature. These usually include abdominal cramps, nausea without vomiting, and diarrhea. Although uncommon, some victims report gastrointesti- nal symptoms without the common vasomotor symptoms (Sapin-Jaloustre, 1957). In severe cases, shock, brochospasms, suffocation, and severe respiratory distress have occurred (Halstead, 1967).

Morbidity values vary from 0.07% to 100% in the literature. Such figures are misleading, as different lots of canned fish may differ greatly in their histamine content. The level of histamine in a whole tuna varies with location, i.e. histamine levels are much higher in the caudal fin area than in the ventral zones (Sapin-Jaloustre, 1957). It is therefore possible for some people to eat fish toxic to others and not be poisoned.

C . CASES OF HISTAMINE TOXICITY

Many cases of scombroid poisoning are thought to be undiagnosed and unreported (Anon., 1973b). Small outbreaks remain undetected even today, as the symptoms are not particularly severe nor long lasting. In mild cases of short duration, medical consultation may not be sought (Foo, 197%). Cases of his-

116 SALLY HUDSON ARNOLD AND W . DUANE. BROWN

tamine poisoning have been reported since the late 1930s and early 1940s. More recently the Japanese performed many comprehensive studies on histamine toxicity and histamine formation in fishery products. The Center for Disease Control (CDC) in Atlanta, Georgia began its food-borne surveillance program in 1966 and most scombroid poisoning events in the United States are now reported through this Center. CDC acknowledges only thirty outbreaks of scombroid type fish poisoning in the United States from 1966 to 1975 (Anon., 1975~). A repre- sentative review of some scombroid poisoning cases follows. Atypical cases are also discussed, such as histamine type poisoning from nonscombroid fish and cases in which no histamine could be detected in the incriminated food.

Legroux er ul. (1946) described two histamine outbreaks from albacore (Thun- nus afulunga) and performed tests which incriminated histamine as the causative factor. The first case occurred in August, 1941 in which 22 of 28 individuals who ate "fresh" tuna showed the usual histamine poisoning symptoms. Guinea pigs could be killed by injections of the tuna homogenate, sterile filtered homogenate, and the heated filtrate. The animals showed symptoms resembling that of histamine shock. The suspension remained toxic after thirty minutes of boiling and after passing on a Chamberland candle, presumably eliminating the possibility of microbial toxins (Sapin-Jaloustre, 1957). Death of the guinea pigs could be prevented by simultaneous injection of neo-antergan, an antihistamine drug. The histamine level was estimated as 1 to 5 gm/kg of flesh (100 to 500 mg%) by assay with the guinea pig ileum test. Legroux er al. (1946) concluded that the cause was not bacterial and that the amount of histidine in the meat could not account for the amount of histamine formed.

Strom and Lindberg (1945) described several cases of histaminelike poisonings in Norway. Fresh tuna (Thunnus rhynnus) and canned tuna were implicated. Symptoms included severe headache, reddening of the body, slight shivering, subnormal temperature, and cardiac palpitations. A toxic substance was isolated from a suspect tuna and identified as histamine. Intravenous injection of the toxic substance killed guinea pigs. Stram and Lindberg proposed that the histidine in the protein of tuna had been decarboxylated to form histamine.

Van Veen and Latuasan ( 1 950) discussed the problem of histamine poisoning associated with the consumption of skipjack tuna (Katsuwonus pelamis) in In- donesia. The fish was harmless if caught and eaten immediately. Tropical temperatures appeared to enhance the poisonous characteristics, although the fish did not appear spoiled. Salted and dried skipjack were more prone to cause poisoning than were the manufactured canned product, possibly due to bacterial growth and subsequent decarboxylation of the histidine. Most toxic samples contained 500 to 700 mg% histamine. Causative bacteria were isolated, but not identified.

Black skipjack (Euthynnus linearus) was responsible for a more neurotoxic type of fish poisoning outbreak on Johnston Island (situated near the Hawaiian Islands) in 1950 (Halstead, 1954). The fish was said to have been eaten on

HIS1 A M I N t (''1 TOXICITY F R O M FISH PRODUCTS I I7

previous occasions without producing any toxic symptoms. Five out of five people consuming the fish developed nausea. comiting. tingling. intestinal cramps. cold clammy skin, and mild diarrhea a few hours after ingestion of the meal. Symptoms subsided after 35 hours with a convalescent period of several weeks, during which time weakness and muscular pains were the typical synip- toms. The Johnston Island outbreak is significant because the ingestion of black skipjack presumably caused symptoms typical of the ciguatera-type (more coni- nionly produced by the reef fishes) rather than symptoms of scombroid poisoning.

Kawabata ef 01. (195Sa) studied the Japanese episodes of allergylike food poisoning caused by dried seasoned saury (Cololuhis suira) and other products. At that time, seafood poisoning of unknown origin accounted for 60 to 70% of all Japanese food poisonings. They differentiated these unknown poisonings from bacterial food poisoning, chemical poisoning, naturally formed toxic substanccs in plants and animals, and allergy or idiosyncrasy. Fourteen outbreaks, involving more than 1000 people were discussed by Kawabala et ul. (l955a); see Table I . Four of the outbreaks were studied epidemiologically. In all cases studied, other vagus stimulants, e.g., saurine, were believed to be present in addition to histamine (saurine is now believed to be identical to histamine; see Section 11,I). "Samma sakuraboshi" was incriminated in many of the Japanese histamine and histaminelike fish poisonings. I t is made by pickling raw mackerel pike (Col- olubis xrira) for twenty to thirty hours in a wheat gluten syrup, then sun dried (Aiso rt al., 1958a). The product is either broiled before consumption or eaten uncooked.

The first outbreak studied by Kawabata ('1 t r l . (1955a) occurred in Hiroshima in I953 after the consumption o f sanima sakuraboshi (Cololubis srriru). All who atc the product became i l l . The incubation period ranged from 30 minutes to 2% hours. Typical symptoms included reddening of the face and upper body, palpitation, severe headache. and nausea. Diarrhea, abdominal pain, and vomiting were absent. All victims recovered. Inorganic poisons. alkaloids and other preservatives were not found in the fond. About 400 to 500 mg% histamine was detected in samples by chemical methods, as opposed to 0.3 to 1 . 1 mg% in the controls. The second outbreak occurred in 1953 in Kumamoto. Japan. Of 850 consumers of a lunch. 85 complained of burning sensations, reddening. flush, palpitation, and headache. Some vomited. others had diarrhea; all body tenipera- tures were normal. The incubation period ranged from 30 minutes to 1 % hours. Canned seasoned mackerel (Scotnbcr .jtrponicws) was incriminated a s thc toxic food. The bacterial count in the cans was negative. color of the meat subnormal with a pH of 6.0 to 6.2 and the meat had a slightly irritative tastc. The low morbidity (10%) was explained because the cans were from different companies and only one sample contained the toxic substance. Another outbreak occurrcd in 1954 in Kanagawa, Japan involving elcvcn people who ate frigate tuna ( A ~ r i s

TABLE I A LIST OF REPORTED SCOMBROID TOXICITY OUTBREAKS IN JAPAN, 1951-1954 BASED ON KAWABATA ET AL. (1955a).

Date Cases Source Symptoms

Oct. 1951

June 1952 Oct. 1952

Oct. 1952

Oct. 1952

Nov. 1952

Feb. 1953

July 1953

Oct. 1953

Oct. 1953

Nov. 1953

Dec. 1953

Aug. 1954

Oct. 1954

700

17 25

22

94

6

I I

85

72

6

3

13

I I

90

samma sakuraboshi (Cololabis suira)

iwashi sakuraboshi samma sakuraboshi

(Cololubis saira) samma sakuraboshi

(Cololabis saira) samma sakuraboshi


(Cololabis suira) samma sakuraboshi

(Cololubis suiru) canned mackerel

(Scomber japonicus) samma sakuraboshi




(Cololabis saira) frigate tuna

(Auxis thaiard) samma sakuraboshi

(Cololubis suiru)

Flushing of the body and itchiness. Some diarrhea

Headache, chills, flushing, rash, and fever Facial flush, headache, nausea, and vomiting. Some

Headache, chills, and facial flush. Some diarrhea and

Headache, chills. and facial flush. No diarrhea or

Headache and chills

and vomiting

diarrhea

vomiting

vomiting

Facial flush, cardiac palpitation, headache, and nausea

Flushing, cardiac palpitation, and headache. Some vomit-

Headache, flushing, rash and vomiting

Headache, chills, and reddening

No vomiting or diarrhea

ing and diarrhea

No diarrhea

Diarrhea and vomiting

Rash

Flushing of the body and facial rash. No headache, fever, diarrhea, or vomiting

Flushing, rash, and headache. Some vomiting and nausea. No diarrhea

1215 (Total cases)

HISTAMINE (‘?) TOXICITY FROM FISH PRODUCTS 1 I9

thazard) which had been caught the previous day. They experienced typical symptoms. Histamine was detected in the fish by paper chromatography, Bac- teriological examination was negative. Histamine levels were 97 to 128 mg%. Volatile basic nitrogen and trimethylamine nitrogen values were normal.

Boyer et al. (1956) studied a large intoxication from four tons of “fresh” tuna (Thunnus thymus). Nearly 500 people were affected, with a morbidity rate of 13%. Nine batches were analyzed with histamine values ranging from 28 to 4000 mg%; various bacteria were present in significant amounts. It was concluded that the amount of available histidine in the tuna could account for the elevated histamine levels and that the histamine was produced by bacterial decarboxylation. Initial contamination presumably occurred because the fish were not prop- erly chilled, nor were the guts removed. The authors proposed close surveillance of sea practices of fisherman, rather than onshore inspection, which was considered useless.

In 1968, eight of nine people became ill 30 minutes after eating “fresh” tuna fish (Anon., 1968). Typical scombroid toxicity symptoms with associated gas- trointestinal symptoms lasted from 1 to 4 hours. Patients were given antihistamines with subsequent marked improvement. The fish contained 425.5 mg% histamine and was negative for Proteus species. In Vermont in 1972, four people experienced nausea, diarrhea, and prostration fifteen minutes to one hour after consumption of “fresh” tuna steak purchased from a local market (Anon., 1972). Histamine formation was attributed to the lengthy unrefrigerated storage (5 1-55 hours) of the tuna at the market. An employee eating 1 Vi pounds of the tuna 3 hours after storage did not get ill. Citrobacter, Proteus, Enterobacter, and Streptococcus were isolated from the remaining tuna in the market. Fresh tuna was again incriminated in New York in 1975 (Anon., 1975c,d).

A family of four experienced headache, nausea, and diarrhea two hours after eating commercially smoked albacore (Thunnus alalunga) in 1972 (Anon., 1973b). Recovery occurred in 3 to 5 hours, except for the father who was hospitalized for shock. Intravenous fluids and antihistamines rapidly improved his condition and he was discharged two days later. No fish could be obtained for chemical analysis, but the outbreak was presumed to be caused by bacterial degradation of histidine to histamine.

In 1973, the Center for Disease Control reported the first recorded outbreak of scombroid fish poisoning from a commercially canned food product in the United States since CDC began food-borne surveillance in 1966 (Anon., 1973d). Canned tuna caused 254 clinical cases in eight states (Anon., 1973a). The symptoms included immediate oral burning and blistering, followed in 30 to 45 minutes by headache, abdominal cramps, diarrhea, and flushing (Merson etal. , 1974). No cases required hospitalization. Nine assays for histamine produced values ranging from 76 to 280 mg% in the incriminated lots, with controls at the 2.7 mg%

120 SALLY HUDSON ARNOLD AND W . DUANE BROWN

level (Anon., 1973d). These lots were later recalled by the Food and Drug Administration.

A case of histamine poisoning was reported in 1973 in which no histamine could be detected in the incriminated food (Anon., 1973~) . Thirty o f48 children experienced a sudden onset of a rash associated with intense itching 15 minutes after beginning lunch. Symptoms lasted 15 minutes to 2% hours. A tuna casserole, made from commercially baked tuna, was incriminated as the source of the illness. The casserole was negative for bacterial histamine formers and contained no detectable histamine. Cans in the remaining cases of the lot appeared normal. Despite the negative findings, the incubation and symptoms of the outbreak were thought to be consistent with those of scombroid fish poisoning.

A number of cases of histaminelike poisoning have been reported in which the incriminated food was not of the scombroid type. Scombroid poisoning was reported as the cause of three incidents in New Zealand in 1973 (Foo, 1975a). Two cases involved a canned mackerel product but the third involved smoked kahawai. This is presumably the first report of scombroid poisoning from kahawai, a nonscombroid fish, although it was assumed that other cases involving kahawai had occurred in the past. A level of 800 mg% histamine was detected in the kahawai samples. The kahawai had been allowed to dry for 3 days before smoking. I t was presumed that bacterial action formed toxins during the drying and that these toxins (histamine and histaminelike compounds) survived the smoking process. Kingfish was later implicated in a histaminelike outbreak in New Zealand (Foo, 1975b).

Scombroid toxicity from mahi-mahi (dolphin fish) was reported in 1973 (Anon., 1973e). The first case involved two women after eating mahi-mahi (Corypharna hippuuus) at a restaurant. Dolphin fish belong to the family Coryphaenidae which is unlike the Scombridae. However, the flesh of dolphin fish does contain large amounts of free histidine (Hibiki and Simidu, 1959). Two subsequent mahi-mahi outbreaks occurred in the state of California later in the year. It was determined that there was improper refrigeration at the time the fish was caught, and also improper handling at the retail level which could result in scombroid poisoning (Anon., 1973e). A subsequent histamine poisoning linked to mahi-mahi occurred in 1975 (Anon., 1975a).

It can be concluded that most histaminelike outbreaks occur after consumption of scombroid fish or other fish normally containing large amounts of free histidine in muscle tissue. The incriminated fish generally contains histamine levels in excess of 100 mg%.

D. EARLIER REVIEWS

Earlier reviews of histamine toxicity from fish products include Sapin- Jaloustre (1957), Kimata (1961). Halstead (1967) and Ienistea (1971, 1973).

HISTAMINL (?) TOXICITY FROM FISH PRODUCTS 121

Douglas (1970. 1975) provides an excellent review of the pharmacology of histamine.

II. MECHANISMS OF FORMATION OF HISTAMINE IN FISH

A. INTRODUCTION

Suzuki et ul. (1909) were among the first to report the presence of histamine in tuna extracts. Geiger et ul. (1944) investigated the content and formation of histamine in mackerel (Scornheromorus concolor), sardines (Surdinops sugux) and albacore (Thunnus ululungu) extracts. Early studies concentrated on histamine formation from autolysis (Geiger et al., 1944; Kimata and Kawai, 19S3e; Hayashi, 1954). although it was concluded that histamine was formed post mortem from bacterial contamination (Geiger ef ul., 1944). Histamine forming bacteria were later isolated and further characterized. Extensive studies of bacterial histidine decarboxylases were subsequently performed.

B . HISTAMINE FORMATION BY “AUTOLYTIC” ENZYMES

Geiger et al. (1944) studied the production of histamine by autolytic enzymes and concluded that bacterial action was responsible for the formation of histamine. A sample of mackerel muscle paste incubated with 3% chloroform and 5% toluene produced an insignificant amount of histamine in 24 hours, whereas a sample without these preservatives produced a considerable amount of histamine. Kimata and Kawai (1953a) arrived at similar conclusions with red meat fish (mackerel and tuna). Small amounts of histamine were thought to be produced by enzymes inherent in the meat. Under optimal autolytic conditions of pH (pH 3 . 5 4 . 5 ) and temperature (40°4S”C) only 10 to IS mg% histamine was formed from frigate tuna (Auxis thuzard) and chub mackerel (Scomber juponicus) (Kimata and Kawai, 1953d,e). White meat fish produced substantially lower levels of histamine by autolysis (Kimata and Kawai, 1953d,f).

It is most likely that the histamine produced by “autolysis” was due to previous bacterial contamination or unsterile conditions during experimentation, thus enabling histamine production by the contaminating microorganisms. Kimata and Kawai (1 953a) acknowledged that their results could have been influenced by the action of enzymes formed from bacteria which had already grown on the fish before the initial experiments. Ferencik ( 1 970) studied strains of Hufniu, Proteus morgunii, and E . c d i that were able to form high levels of histamine in sterile tuna and skipjack flesh. Histamine formation depended on the free histidine content of the fish and the histidine decarboxylase activity of the bacteria. Fish flesh experimentally infected with bacteria having no active his-

122 SALLY HUDSON ARNOLD A N D W . DUANE BROWN

tidine decarboxylase did not contain histamine at any stage of decomposition. Flesh kept under sterile conditions also failed to form any histamine.

C. DETECTION OF BACTERIAL HISTIDINE DECARBOXYLASES

It is not the purpose of this review to provide a comprehensive discussion of the detection of bacterial histidine decarboxylases, but a brief overview has been included. Histamine has often been detected in bacterial cultures and fish infu- sions by the conventional chemical and biological methods described in Section 111. However, some methods have been developed specifically for detecting, measuring, and characterizing bacterial amino acid decarboxylases.

Eggerth et al. (1939) developed an improved extraction method for measuring small amounts of histamine in bacterial culture media. Gale (1940, 1946) studied bacterial amino acid decarboxylases in detail by measuring the evolution of C 0 2 with a Warburg manometer. Epps (1945) extracted and purified L-histidine decarboxylase from Clostridium welchii (now called C. perfringens) and examined its properties. Several cell free amino acid decarboxylases were made and characterized by Gale ( 1 946). Histidine decarboxylase from Lactobacillus 30a was purified by Rosenthaler et al. (1965); its substrate specificity, sterospecificity, composition, and subunit structure were studied by Chang and Snell (1968a,b).

Mpller (1954a,b) developed several methods for activity determination and distribution of amino acid decarboxylases in enteric bacteria. A method for measuring the total amino acid decarboxylase contents of bacteria was devised (Mpller, 1954a). Mpller (1954b, 1955) also developed a color test for measuring various amino acid decarboxylases which was subsequently modified by Shaw and Clarke (1955). The color test for decarboxylases was later shown to be an indicator of pH change in the medium and not necessarily specific for the presence of histidine decarboxylase enzymes (Havelka, 1969). Levine and Watts (1 966) developed a radioactive method for measuring histidine decarboxylase activity. Histidine was labeled with 14C in the carboxyl carbon and the evolved [14C]C02 was trapped and measured. Schayer (1971) provides a recent review on the determination of histidine decarboxylase activity.

D. BACTERIA RESPONSIBLE FOR HISTAMINE FORMATION

Early microbial studies indicated that many bacteria were able to form amino acid decarboxylases. Eggerth (1939) studied the histidine decarboxylase activity of Escherichia, Aerobacter (now called Klebsiella), Salmonella, and Shigella. Gale (1 946) detected histidine decarboxylase activity in Escherichia, Clos- tridium, and Klebsiella but not in Proteus, Bacillus, or Streptococcus. Ehris- mann and Werle (1948) found that Mbrio, Proteus, and other gram-negative bacteria were able to form histamine.

HISTAMINE ( ? ) TOXICITY FROM FISH PRODUCTS 123

Van Veen and Latuasan (1950) isolated two strains of anaerobic salt tolerant bacteria able to produce 35 mg of histamine per 10 gm of fish flesh (350 mg%) from skipjack tuna (Karsuwonus pelarnis) indicated in a histaminelike outbreak of poisoning. They were identified only as a small gram-positive coccus and a gram-positive rod.

Kimata and Kawai (1953b) isolated a “new” species of bacteria able to produce large amounts of histamine from spoiled fish. It was given the name “Achromobacter histarnineurn, ” although it was later shown to be identical to Proreus rnorganii (Kimata et al., 1958; Kimata and Kawai, 1958). Production of histamine by “ A . histarnineurn” corresponded to the growth of the organism when incubated at different temperatures (Kimata and Kawai, 1953~) . When a frigate tuna (Auxis thazard) extract was inoculated with the organism, more than 200 mg% of histamine was formed at 27°C in three days.

Kimata and Akamatsu (1955a) later reported that two strains of “ A . histarnineurn,” known as Type 1 and Type 2, existed. Both types were able to produce large amounts of histamine from histidine and only small amounts of ammonia from amino acids. It is most probable that the two “types” were merely different strains of Proteus rnorganii, or perhaps different Proteus species. Growth and histamine formation of the two types were extensively studied (Kimata and Akamatsu, 1955b). Type 2 had a growth optimum of 30°C; Type 1 had an optimum of 20 to 25°C. Production of histamine at various temperatures varied with the growth rate of Type 1, but not with Type 2. The pH optimum for histamine formation for Type 1 was pH 5 and that of Type 2 was pH 6 . At a pH of 7, Type 1 produced very little histamine, whereas Type 2 was able to produce a significant amount. The optimum sodium chloride concentration for growth was 1% and for histamine production was 2 to 3% in both types.

Proteus organisms continued to be implicated as the responsible histamine formers in scombroid and other fish. Kawabata et al. (195613) isolated 78 strains of bacteria from “sashimi” (sliced raw fish made from Parathunnus rnebachi) that had previously caused a histamine outbreak. Eleven of the 78 strains isolated from the sashimi were able to produce histamine. Five were identified as Proteus vulgaris, three as Proteus rnirabilis and three as Proteus morganii. They suggested a mode of putrefaction in which the organism could produce large amounts of the toxic substances without producing signs of deterioration in the food. Kawabata and Suzuki (1959b) reported that histidine decarboxylase activity was highest when cell division ceased, although slight activity was still detectable when the bacteria reached the death phase.

Aiso et al. (1958b) studied the histidine decarboxylase activity in 84 strains of Morganella (now called Proteus morganii). Histidine decarboxylase activity was measured by the quantitative determination of histamine formed in the culture media and by estimation of the QCOz by the Warburg method. All Proreus morganii strains, irrespective of source, were able to produce histamine. A


maximum of 400 mg% histamine was formed in fish meat infusion broth within 76 hours. Escherichia, Shigella, Protrus vulgaris, and Clostridium perfringens also exhibited slight histamine forming activity. Similarly, others (Kimataer al., 1960) tested 88 Proteus strains. All Proteus morganii strains could produce histamine; P . vulgaris, P . mirabilis, and P . rettgeri produced either insignificant amounts of histamine or none.

The presence of high levels of Proteus organisms is currently used as a criterion of scombroid poisoning (Anon., 1972, 1975c,d). Other organisms such as Hafnia and E . coli are able to produce significant amounts of histamine, but at much slower rates (Ferencik, 1970). Proteus morganii is especially incriminated as the responsible histamine former because of its ability to rapidly produce histamine in excess of 100 mg%.

E. OCCURRENCE OF HISTAMINE FORMERS

Studies of the bacteria involved with fish spoilage indicated that only a small percentage were able to produce large amounts of histamine. Kawai (1962) indicated that histamine-forming bacteria isolated from fresh fish were found much less frequently than those organisms able to produce other amines.

Kimata and Kawai ( 1 953g) isolated one strain out of twenty-five that was able to produce histamine in significant quantities. The responsible organism was identified as “Achromobacter histamineum” (Proteus morganii). When fresh mackerel (Scomber japonicus) were spoiled at 20°C, 5 to 30% of the total number of bacteria were histamine formers (Kimata and Tanaka, 1954a). Produc- tion of histamine was again concluded to be the result of a certain microorganism ( P . morganii) and not due to the action of all kinds of bacteria causing spoilage.

Histamine formers have been detected as part of the normal surface microflora of fresh fish. However, Cantoni et a/. (1976a) reported that bacteria such as Proteus, Escherichia, and Clostridium were contaminants and not part of the normal microflora of fish. Kimata and Tanaka (1954b) found that 0.1 to I % of the total surface bacteria were usually histamine formers, whereas ammonia formers accounted for 10%. Live fish generally contained higher percentage levels of histamine formers than “fresh” (marketed) fish. The actual number of histamine formers did not decrease; the nonhistamine formers grew more rapidly and consequently the percentage level of histamine formers was lower in the marketed fish.

F. FREE HISTIDINE AS A HISTAMINE PRECURSOR

Geiger (1944b) observed that the ability of E . coli to decarboxylate histidine was highly specific since histamine formation was inhibited by acylation of the amino group of histidine. Geiger (1948) later studied histamine formation with a Clostridium welchii (now called C. perfringens) enzyme preparation, a strain of

HISTAMINt (''1 TOXICITY FROM FISH PRODUCTS 125

E . coli. and a strain of marine bacteria. All were able to form histamine from free L-histidine, but none was able to form histamine from aspartyl-histidine o r histidyl-histidine. It was concluded that peptide linkages to either the -COOH or -NH, group of histidine prevented bacterial decarboxylation.

It remains well documented that free histidine is required for histamine formation. Ferencik (1970) has recently concluded that the main condition for histamine synthesis was an adequate concentration of free histidine in the fish, as only free histidine can be decarboxylated. Toxic levels of histamine are generally limited to the red meat of free-swimming species because these species contain a large amount of free histidine in the muscle (Aiso et a l . , 1958a; Lukton and Olcott, 1958) while crustacea and white meat fish contain very low levels of histidine (Hibiki and Simidu, 1959; Lukton and Olcott, 1958).

G. HISTIDlNE DECARBOXYLASE

I . Effect of Trmperature

Gale ( 1940) observed that bacterial amino acid decarboxylases were generally most active at temperatures below 30°C. Kimata and Kawai (1953a.c) reported that Achromobacter histarnitieum (now called Proteus morganii) had an optimum growth temperature of 20" to 25"C, but produced the highest levels of histamine at 20°C. Negligible amounts of histamine were formed at 35°C. No histamine was produced by the organism at 40°C (Kimata and Akamatsu, 1955b) since growth could not occur at that temperature. Hibiki and Simidu (1959) later found that the responsible histamine-forming bacteria were killed at 60°C.

Wide variation in histamine formation can commonly be found in storage trials at ambient temperatures. Edmunds and Eitenmiller (1975) recently observed that histamine production varied significantly between storage trials performed at ambient temperatures. Omura ( 1976) detected one hundredfold variations in histamine concentrations from skipjack tuna that were allowed to spoil under similar conditions. Kimata and Kawai (1953a) allowed mackerel fillets to undergo bacterial spoilage at various temperatures. The amounts of histamine present at the time in which the fish were judged organoleptically unacceptable as food was as t'ollows:

Storage temperature Histamine Time fish judged unacceptable

35°C 14 mp% 20 hr 17°C 354 mgL% 75 h r

6-7°C 50-70 mg% 150-200 hr

A similar study with whole fish (Kimata and Kawai, 1953a) indicated that at the time in which the fish were judged unacceptable (46 hours), 826 mg% histamine had been produced at 23°C. In contrast, others (Anon., 1975b) studied the


formation of histamine in mackerel under various storage conditions and concluded that the fish were unfit for commercial use by the time the histamine concentration reached 100 mg%.

Numerous studies have been made on histamine production at near-freezing and freezing temperatures. Shewan and Liston (1955) reported that histidine was easily decarboxylated at 0°C. It has been suggested (Ota and Kaneko, 1958; Kalyani and Bai, 1965; Edmunds and Eitenmiller, 1975) that histamine-forming bacteria were killed or unable to produce histamine at this temperature. Kalyani and Bai (1965) observed that histamine production was slowed at 10°C and nearly terminated at 5°C due to the destruction of histamine-producing bacteria. Edmunds and Eitenmiller (1975) found that little histamine was formed at 4°C in storage trials of Spanish mackerel and other fish. They concluded that the psy- chrophilic microorganisms did not readily decarboxylate free histidine. Others (Anon., 1975b) reported that small amounts of histamine were formed at 6"C, depending on the humidity of the environment and that no histamine was formed at -20°C in two months. Current work in progress has shown that certain strains of P . morganii and P . vulgaris are able to produce levels of histamine in excess of 120 mg% when allowed to grow in tuna infusion broth for six days at 7°C. No histamine was formed by P . morganii, P . vulgaris, or Hafnia strains after one months incubation at 1°C.

It is evident that other factors besides temperature and time can drastically influence histamine formation. Possible factors may be related to the original bacterial flora of the fish, prior bacterial contamination from catching and handling the fish, environmental factors, etc. However, it can be concluded that one of the best methods for retarding bacterial histamine formation in fish is to store the fish at temperatures below the freezing point of fish muscle.

2 . Effect of pH

Early studies of amino acid decarboxylases by Gale (1940, 1946) and Epps (1945) demonstrated that the pH optimum was acidic, ranging from pH 2.5 to 6.5. Alin (1950) concluded that during bacterial growth an acid medium stimulated the formation of decarboxylases. Aiso et al. (1958b) found that Morganella (now called Proteus morganii) was able to form histidine decarboxylase in both neutral and acidic environments. Thirteen Proteus strains, including P . morganii, P . mirabilis, P . rettgeri, and P . vulgaris were shown to have a histidine decarboxylase pH optimum of 6.0 to 6.5 (Kawabata and Suzuki, 1959a). Kawabata and Suzuki (1959b) concluded that the maximum production of histamine in Proreus morganii occurred at pH 5.1, the lowest value supporting growth. Organisms grown at pH 5 . 5 to 7.3 possessed about 50% of the maximum activity and those grown at pH 7.6 to 8.7 possessed 10% of the maximum activity. Simidu and Hibiki (1954b,c,d) found that the pH of fresh scombroid

HISTAMINE (? ) TOXICITY FROM FISH PRODUCTS 127

fish flesh generally ranged from pH 5.5 to 6.5 . The slightly acidic pH of the fish thereby enhances the production of histamine by the responsible histidine decarboxylating bacteria.

3 . Effect of Carbohydrates

Early workers (Eggerth, 1939; Gale, 1940) observed that glucose and other fermentable sugars intensified bacterial amino acid decarboxylase activity.

Aiso et al. (1955) noted that the addition of glucose to diluted fish meat extracts caused a substantial increase in histamine formation. Kimata and Kawai (1958) found that carbohydrates such as glucose and fructose enhanced the formation of histamine in diluted fish extracts but not in synthetic media. The synthetic media contained sufficient amounts of carbohydrate whereas the diluted fish extract was slightly deficient. They concluded that the bacteria required a certain amount of carbohydrate as an energy source for biosynthesis and enzyme production. Others (Kawabata and Suzuki, I959b) observed that the presence of a fermentable carbohydrate, such as glucose, enhanced both growth and histidine decarboxylase activity in Proteus morganii. Glucose concentrations of 0.5 to 2% were most effective in mackerel infusion media. Levels in excess of 3% inhibited enzyme formation despite the low pH produced during bacterial growth on glucose.

4 . Effect of Vitamins and Coenzymes

Several studies of bacterial histidine decarboxylase have indicated that the addition of vitamins and coenzymes did not enhance histamine formation. Epps (1 945) reported that codecarboxylase was absent in L-histidine decarboxylase extracted from Clostridium welchii (now called Clostridium perfringens). Gale ( I 946) observed that the addition of cofactors such as pyridoxine (vitamin Be) and nicotinic acid (vitamin B3) in excess of simple growth requirements sometimes promoted tyrosine, lysine, and ornithine decarboxylase but not histidine decarboxylase. Rosenthaler et al. (1965) determined that histidine decarboxylase from Lacrobacillus 30a did not require pyridoxal-5'-phosphate as a coenzyme. They concluded that no known coenzyme was required. Work with Proteus morganii by Kimata and Kawai (1958) showed that none of the vitamin B complex promoted histamine formation in synthetic or fish infusion media.

5 . Effect of 0,yygen Tension

Kimata and Kawai (1958) found that the addition of cysteine, cysteine, and methionine stimulated histamine formation in P . morganii. Other amino acids (except histidine, of course) had no such effect. They suggested that cysteine,

128 SALLY HUDSON ARNOLD AND W. DUANE BROWN

cystine, and methionine effectively reduced the redox potential of the media, thereby stimulating histamine production. Sodium-thioglycollate and ascorbic acid (redox potential reducers) also stimulated histamine production. Kimita and Kawai (1959) later observed that histamine formation in washed cell suspensions of P. morganii were affected by the oxygen tension. Histamine formation in aerated cultures, achieved by the addition of bubbled gas, was much less than that from anaerobic and aerobic cultures. They (Kimata and Kawai, 1959) concluded that the histidine decarboxylase activity of P . morganii was destroyed or inactivated in the presence of oxygen. Ferencik (1970) reported that anaerobic conditions caused slower histamine formation than aerobic conditions in Hafnia.

H. BACTERIAL DESTRUCTION OF HISTAMINE

Gale (1 942) reported that any study of bacterial amine production should also take into account any further breakdown of these substances by other microorganisms. Ienistea (1971) suggested that bacterial histaminase could play an important role in foods containing high concentrations of histamine. It was proposed that an equilibrium between histamine production and destruction occurred in these foods. [Note: Histaminase refers to the enzyme capable of oxidative deamination of histamine and is frequently called diaminoxidase (Douglas, 1 9 7 5 ) ~

Bacterial histaminase (diaminoxidase) activity has been detected in several types of bacteria including some species of Pseudomanas (Werle, 1940. 194 1 ; Gale, 1942; A h , 1950), Profeus (Werle, 1940), Escherichia (Werle, 1941; A h , 1950), Vibrio (Ehrismann and Werle, 1948), Clostridium (Ehrismann and Werle, 1948). and Klebsiella (Ehrismann and Werle, 1948). Gale (1942) reported that the bacterial enzymes capable of histamine oxidation were inducible. Bacterial histaminases were best produced under somewhat alkaline conditions (pH 7.5-8), although moderate histaminase activity was detected under slightly acidic conditions (Gale, 1942; A h , 1950).

Ferencik (1970) reported that a strain of Proteus morganii, after being inoculated into a sterile tuna flesh homogenate, produced large amounts of histamine. However, a significant amount of the histamine was soon decomposed by the organism. A subsequent sterile addition of histidine to the inoculated homogenate again resulted in histamine formation, then histamine destruction. Ferencik (1970) concluded that the histamine production and destruction by the P. morganii strain was determined by the concentration of free histidine in the fish homogenate. The minimum histidine concentration required for histidine decarboxylase activity appeared to be 100 to 200 mg%. Work in progress (Arnold, 1976) deals with a similar observation in a P. morganii strain and a P. vulgaris strain previously isolated from intentionally spoiled skipjack tuna.


I . “SAURINE”

Kawabataet ul. (1955a) suspected that an additional toxin or a compound able to act synergistically with histamine was present in foods causing histamine poisoning. Chemical methods often produced histamine values ten times less than that found by the guinea pig ileum test, suggesting that an unknown vagus stimulant was present. [Note: Vagus refers to the tenth pair of cranial nerves. Among other functions, these nerves innervate the muscles of the abdominal viscera and conduct the impulses to the brain.] A newly isolated vagus stimulant was detected by one- and two-dimensional chromatography (Kawabata et ul., 1955b). I t was named “saurine.” referring to the fish saury (Cololabis suira) that had often been implicated in histamine poisonings in Japan. Proteus rnorganii was later incriminated as the source of saurine (Kawabata rt d., 1956b). as i t appeared to be able to produce large amounts of both histamine and saurine. Saurine was differentiated from histamine by its lower Rf value, its negative reaction with diazo reagent, and its possible insolubility in alcohol (Kawabata et al., 1955b). Saurine was characterized as a heat stable, basic, low molecular weight substance (Kawabata ef ul., 195Sb). Its chemical and biological effects were similar to those of histamine; saurine exhibited an additive and not a synergistic effect to that of histamine (Kawabata et nl., 1955~) . The effects of antihistamines on saurine were proportional to their antihistaminic activity (Kawabata et ul., 1955d).

Olcott and Lukton (1961) suggested that “saurine” could be an artifact. A culture of Proteus morguriii previously able to produce saurine was obtained from Kawabata. A tuna extract was inoculated with the organism. Two spots were obtained by two-dimensional chromatography, both able to exhibit vagus- stimulating activity on the guinea pig ileum. When histamine alone was added to the original tuna extract, a similar pattern was obtained, suggesting that histamine was able to produce both spots under these conditions. Kawabata subsequently informed Olcott and Lukton that the culture had lost its ability to form saurine (Olcott and Lukton, 1961).

Work in the 1950’s demonstrated that amines could produce more than one chromatographic spot in some situations. Waldron-Edward (1954) observed that amines were able to produce multiple spots on paper chromatograms under certain conditions and that typical color reactions (e.g. the ninhydrin reaction) could be adversely affected. West and Riley (1954) reported that two sharply defined spots corresponding to histamine appeared on chromatograms of tissue histamine. Both spots produced vagus-stimulating activity on the isolated guinea pig ileum.

There is little doubt that ”saurine” is identical to histamine. Unfortunately, the presence of saurine has been discussed in the literature since its “discovery” and it continues to be mentioned (Anon.. 1973d; Foo. 1975a).


J . CONCLUSION

Bacterial histamine formation is dependent on (1) an adequate concentration of histidine in the free form, (2) the presence of microorganisms able to produce histidine decarboxylase, and (3) conditions conducive to histidine decarboxylase synthesis and subsequent decarboxylation. Scombroid fish contain both a high level of histidine in the free form and a significant number of histamine-forming bacteria as part of their natural microflora. It is therefore necessary to maintain conditions that adequately supresses histamine formation; the most practical method is to keep the fish at temperatures below freezing.

111. DETECTION AND DETERMINATION OF LEVELS OF HISTAMINE IN FISH

A. GUINEA PIG ILEUM CONTRACTION

The classical method for the determination of histamine is based on the fact that it will cause contraction of guinea pig ileum. Such methods currently employed are based on the procedures suggested by Barsoum and Gaddum (1935). These authors actually proposed that response of two tissues, guinea pig ileum and rectal cecum of a fowl, be employed. In their opinion, if a test material applied to both tissues caused contraction in both instances it could be considered to be histamine without further confirmation. Subsequently, however, most workers have employed the guinea pig tissue alone.

The first application of this method to the analysis of histamine in fish tissue appears to be that reported by Geiger (1944a). He and fellow workers had earlier identified a biologically active substance in marine fish as histamine (Geiger et al., 1944). He detailed how the method could be applied to fish and reported findings on histamine levels in raw and canned sardines and mackerel. He pointed out that canning of these fish did not interfere with subsequent analysis for histamine, and demonstrated the practical application of histamine determination in canned tuna. Hillig (1956) subsequently applied Geiger’s method to several species of tuna at varying stages of decomposition while Sager and Horwiti (1957) compared the bioassay with a chemical method based on coupling histamine, in a chromatographically purified extract from fish, with a diazonium compound. Formation of the latter substance was measured spec- trophotometrically. They found that values with the bioassay were usually higher than those obtained by the chemical method. However, when they slightly modified the extraction procedure used in the bioassay, they were able to obtain results with the modified bioassay that checked well with those obtained by the chemical method.

HISTAMINE ( '?I TOXICITY FROM FISH PRODUCTS 131

Following the description of the use of guinea pig ileum contraction as a bioassay for histamine, there was considerable related work, much of it aimed at improvement in the extraction procedure. One early suggestion was the use of butanol extraction of an aqueous histamine-containing extract, followed by removal of histamine from the butanol by cotton acid succinate (McIntire ef al., 1947). Kadota and Inoue (1953) subsequently applied McIntire's method to the determination of histamine found in decomposed fish. They found the method suitable for rapid determination of histamine in a large number of samples. Other suggestions have included the utilization of various cation exchange resins to purify histamine from tissue extracts (Roberts and Adam, 1950; Michaelson and Coffman, 1969). Work to I956 is well summarized in a review by Code and McIntire (1 956) dealing with the quantitative determination of histamine; this review includes a detailed description of the guinea pig intestine assay as well as a summary of chemical methods developed at that time.

B . OTHER BIOASSAYS

The use of the guinea pig ileum contraction technique is far and away the most commonly applied bioassay. However, others have been suggested. De Waart et al. (1972) evaluated a total of 32 biological test systems, including 6 protozoa, 4 fish species, 5 insects, bull spermatozoa, 4 cell lines, 9 microorganisms, em- bryonated hen's eggs. Daphnia and Artemia for sensitivity to a variety of microbial toxins and histamine. Some of the protozoa, fish, Artemia, and particularly Daphnia were found to be sensitive to histamine. The latter organism, a small fresh-water crustacean, is being used presently by an investigator at the Univer- sity of California at Davis (Blonz, 1976). He has found that the addition of histamine, or extracts from tuna known to have caused human illness, to water housing Daphnia causes the death of 90 to 100% of the animals in 20 to 50 minutes, compared to a 0 4 % effect in animals receiving an extract from good tuna. Daphnia are readily available and easy to house, with algae being a suitable food source. Potential significance of their use at this time awaits additional experimental findings.

C. FLUOROMETRIC ASSAY

In spite of the fact that accurate measurements are known to be possible with the commonly used guinea pig ileum bioassay, such methods have numerous disadvantages. Provisions must be made for the care and handling of guinea pigs and there is the frequently noted individual variability in ileum response. For these and other reasons, there has been increased interest in the development of suitable chemical means for histamine determination.

The use of fluorometry has evolved as a major tool for the assay of histamine.


A relatively simple fluorometric method, said to be precise and sensitive for histamine, was reported by Shore er al. (1959). It involves extraction of histamine from alkalinized perchloric acid tissue extracts with n-butanol, and its ultimate condensation with o-phthalaldehyde to yield a stable and strongly fluorescing product. The authors noted that levels of histamine as low as 0.005 pg/ml could be assayed. However methods with extraordinary sensitivity are not usually required for assay of histamine in fish, inasmuch as histamine levels in decomposed fish will be much greater than those found in ordinary tissues.

Fluorometric assays have found broad application for the determination of histamine in a variety of biological materials (Noah and Brand, 1963; von Red- lick and Click, 1965; Huffrt al., 1966) and wine (Ough, 1971). There is detailed description of the use of the fluorometric method for the measurement of histamine in a review by Shore (1971), which includes coverage of means of extraction of histamine from tissues together with a description of special purification procedures.

More recent developments in this field include development of the use of fluorescamine to react with amino acids, peptides, proteins, and primary amines (Udenfriend el at., 1972). This reagent reacts rapidly at room temperature in aqueous media and yields highly fluorescent products. The reagent and its degradation products are nonfluorescent. It can detect arnines in the picomole range; again, however, as a practical matter, such limit of detection is not critical for assay of histamine iii fish, where levels are much higher.

Hdkanson and Ronnberg (1974) have suggested an improvement in the fluorometric histamine assay involving condensation with o-phthalaldehyde at -20°C. Among other advantages claimed by reaction in the frozen state is that spermidine, which may interfere in the conventional assay, gives no fluorescence under these conditions. Still another recent modification of an o-phthalaldehyde fluorometric procedure has been proposed, this one involving the use of an IRC-50 column for purifying histamine from biological materials (Cantoni et al., 1976b).

Yamada and Wakabayashi (1974) compared colorimetry, fluorometry , and bioassay for measuring the increase in blood histamine following administration of certain antibiotics. They report that fluorometry and bioassay were superior to the colorimetric determination and that the fluorometric procedure was the most simple of the three.

At the time of this writing, the U.S. Food and Drug Administration is working on the development of a simpler, but still accurate, fluorometric assay for histamine in fish. The method has not yet been published, although an abstract has appeared (Staruszkiewicz et al., 1975); this method has been adopted as an official first action method by the AOAC (Staruszkiewicz, 1977).

Another new method for the determination of histamine in tuna fish by

HISTAMINE ('3 TOXICITY FROM FISH PRODUCTS 133

fluoronietry has been developed (Lerke and Bell, 1976). Histamine is recovered from fish extracts by ion exchange chromatography and then derivatizecl with o-phthalaldehyde. From filtered crude extracts, 12 samples can be determined per man hour including reconditioning of the ion exchange columns. Recoveries of histamine added to extracts from acceptable quality fish ranged from 98- 103%. and recoveries of 94-101% were demonstrated for histamine added to extracts of decomposed fish. The method is claimed to be as accurate BS the official AOAC procedure (see Section 1II.E) and much simpler.

D. GAS-LIQUID CHROMATOGRAPHY

Histamine can be determined by gas-liquid chromatography. For example, Fales and Pisano (1962) described a procedure employing a column containing 4% SE-30 siloxane polymer as the liquid phase. Navert (1975) has described gas-liquid chromatography procedure which is said to separate adequately histidine, histamine, and the naturally occurring methyl histamines. In spite of the possibilities for application of these techniques. they have not achieved widespread use among investigators studying the histamine toxicity problem.

E. COLORIMETRIC ASSAY

Early chemical methods used for the quantitative determination of histamine involved either coupling of histamine with a diazotized aromatic amine to produce an azo-dye, or the reaction of histamine with 2.4-dinitrofluorobenzene (DNFB). Code and Mclntire (1956) discuss both methods in detail, based on knowledge at hand at the time of their review. They conclude that DNFB is the better reagent of the two in that it yields a much more stable colored histamine derivative, is more sensitive. and is potentially more specific than the azo-dye method.

Subsequent to the report of Code and Mclntire. there have been additional studies on colorimetric assays. We have previously cited work in which chemical assay of histamine by diazonium reaction was compared with guinea pig ileum bioassay (Sager and Horwitz, 1957). Ota (1958a.b) has suggested a modification of the azo-dye method based on extraction o f the histamine azo compound with various organic solvents. The procedure resulted in the removal of interfering substances, including histidine.

Tsuda et al. (1959) later questioned Ota's procedure and suggested that there was better removal of interfering substances with ion-exchange chromatography. Weissback et al. (1958) reports a method designed specifically for measuring both histamine and serotonin in the same extract. After protein precipitation, both compounds were extracted with butanol, and the butanol was then passed


through a cotton-acid succinate column, which separated histamine from serotonin. The latter substance was assayed fluorometrically, while the histamine was reacted with DNFB for colorimetric measurement.

Kawabata and associates (1960) reported a simple and rapid diazo method for the determination of histamine, doing their work with fish samples. They used a cationic exchange resin for separating histamine from interfering substances, including histidine, in a trichloracetic acid extract of fish flesh. Aliquots of column eluates were coupled with Pauly’s diazo reagent and the absorbance at 5 10 nm determined by spectrophotometry. They obtained recoveries of added histamine of 99 to 101%. Their column had a very high exchange capacity for histamine, and the presence of histidine, tyrosine, or tyramine in trichloractic acid extracts did not interfere with histamine values. This method, or modifications of it, appears to have been the one most frequently employed by investigators and industry personnel concerned with recent outbreak of toxicity from tuna.

The “official” method for histamine determination, i.e., that detailed in the “Official Methods of Analysis of the Association of Official Analytical Chemists” also employs reaction with a diazonium compound (Horowitz, 1975). However, in the hands of workers in our laboratory and elsewhere, it is tedious and time consuming and could not be considered practical for routine analysis of large numbers of samples. For example, the time required for a single determination, not including sample extraction, may range up to 2 hours.

Obviously, then, there is need for better methodology; hence the work in this direction cited earlier (Staruszkiewicz et al . , 1975; Lerke and Bell, 1976) and by others to be cited later in this review.

F. ENZYMATIC ISOTOPIC ASSAY

A somewhat novel means for the determination of histamine has been proposed by Snyder et d. (1966). Tissue samples were incubated with tracer amounts of [3H] histamine and [14C]-S-adenosylmethionine in the presence of the enzyme histamine methyl transferase. This enzyme methylates histamine, and [3H] [14C] methylhistamine is the only labeled product extracted. The ratio of [ 14C]/[1H] is directly proportional to the amount of unlabeled histamine present in the incubation mixture. The method is claimed to be sensitive, specific, and relatively simple to perform.

G. THIN-LAYER CHROMATOGRAPHY

Schwartzman (1973) described a thin-layer chromatographic method for the separation and quantitat ion of histamine, methy lhistarnine, acetylhistamine, methylimidazoleacetic acid, and imidazoleacetic acid. He used a butanol-glacial

HISTAMINE (?) TOXICITY FROM FISH PRODUCTS 135

acetic acid-water system, and produced fluorescent compounds of the five amines which were quantiated by absorption or fluorescence. Schwartzman and Halliwell (1975) later described application of a thin-layer chromatographic assay to determination of histamine and its metabolities in urine.

More recently, workers interested in the problem of scombroid toxicity have developed two methods that also utilize thin-layer chromatography. Both are claimed to be considerably more rapid to accomplish than those suggested by Schwartzman (1973) and Schwartzman and Halliwell(l975). In one of these new methods (Lin et uf., 1976) ground and mixed fish is extracted with methanol, the extract is filtered and subjected to thin-layer chromatography. Plates are developed with MeOH:NH40H for 70 minutes, dried for 8 minutes, and the histamine spot is developed with ninhydrin. The amount of histamine present is determined by densitometry . This method does not require preliminary column purification of the extract and is a quick and simple method for determination of histamine in fish.

In the second new method, collaborators working on the same problem have employed a technique in which samples of press juice or fish flesh are applied directly to thin-layer chromatography plates (Schutz et al., 1976). The plates are then developed with an acetone-ammonium hydroxide solution, and spots are visualized with ninhydrin or Pauly’s reagent. Chromatographic separation of histamine from other fish components, including histidine, is said to be readily achieved. The method is semiquantitative, but should be quite suitable for routine screening of large numbers of samples. The authors report that histamine levels as low as 2.5 mg% in fish samples and standard solutions are readily detected.

IV. RELATIONSHIP OF SPOILAGE TO HISTAMINE FORMATION

It appears to be generally accepted that the production of histamine is in itself of bacterial origin and therefore does represent a criterion of spoilage or deterioration in fishery products. However, the production of histamine per se would not be expected to be detectable by simple means, e.g., change in appearance or production of off-odors. Fish containing large amounts of histamine may have a normal appearance and odor (Sapin-Jaloustre, 1957). Thus, it is of interest to note any correlation of histamine production with other more commonly used indicators of spoilage or loss of quality in fish. While a great many methods for measuring quality deterioration in fish have been employed over the past few decades, few, if any, have received any sort of widespread acceptance. Some type of sensory evaluation may well be the method of choice, e.g., detection of off-odor, appearance of surface slime, and changes in appearance of gill tissue or eyes. Objective methods frequently suggested include the determination of total


volatile bases or acids, volatile reducing substances, the increase in concentration of hypoxanthine associated with postmortem breakdown of tissue nucleotides (enzymatic or bacterial in origin), the production of ammonia, and, at least in several species, the production of trimethylamine (a substance with a pronounced fishy odor) by bacterial reduction of trimethylamine oxide. The last problem is of less importance with tuna than with many other species of fish. This is not the appropriate place to review the application of these and other methods for evaluation of fish spoilage. A recent book by Connell (1975) offers a useful summary of this topic and of related issues.

In the 1950s, Kimata and associates published a series of papers dealing with the freshness of fish and the amount of histamine present. Their early work dealt with factors such as the effects of pH and temperature on the amount of histamine produced in “red meat” fish such as mackerel (Kimata and Kawai, 1953a,d). They later reported that histamine was produced after the production of ammonia and amino nitrogen had begun during spoilage of “white meat” fish, e.g. rockfish (Kimataet ul., 1954a). They pointed out in the paperjust cited that more histamine was produced during spoilage of mackerel species than that of rockfish. They reported similar findings with spoilage of squid and octopus, i.e. ammonia production preceded that of histamine (Kimata er al., 1954b.c). As would be expected, in every instance they noted that spoilage, ammonia production, and histamine production were enhanced at elevated storage temperatures.

[Note: Some commonly used terms can prove to be confusing in this discussion. The terms “white meat” or “red meat” fish are based on the general surface appearance of the fish, i.e., the degree of redness. Thus, for example, cod species would all be “white meat” fish, while mackerel species would be “redmeat” fish. On the other hand, the terms “red” (or “blood”) and “white” muscle refer to muscle types within an individual fish, “red” muscles being those containing predominantly red fiber types and “white” muscles being those containing predominantly white fiber types. All scombroid fish contain both muscle types in varying amounts, depending on species. In the commercial processing of tuna, only white muscle is canned for human consumption, the red muscle being used primarily for pet food.]

Other Japanese workers have investigated different aspects of the relationship of spoilage to histamine production. Many scombroid fish contain both red (“blood”) and white muscle. In studies of three such species (mackerel, yellow- tail, and bonito) Simidu and Hibiki (1954a) found that the decrease of histidine and the consequent increase in histamine was far lower in “blood” muscle than in white muscle, They also reported that trimethylamine was produced before ammonia, and that the amounts of volatile base and trimethylamine were much larger in “blood” muscle than in white muscle. In other work they found that histamine was produced during early stages of spoilage in mackerel and suggest that spoilage at that state would be difficult to discern (Simidu and Hibiki,

HISTAMINE ( ‘ ? I 70XICITY FROM FISH PRODUCTS 137

1954b). In agreement with others. they found very little histamine produced during spoilage of ‘*white” fish, squid and shrimp although large amounts of volatile base are produced (Simidu et a/ . , 1955). Similar results were obtained with crab (Simidu and Hibiki, 1954~) . I t was subsequently proposed by Simidu and Hibiki (1955a) that volatile bases and their precursors, such as triinethylamine oxide. urea. and free monoamino acids. inhibit the formation of histamine from histidine.

Kalyani and Bai (1965) reported on histamine formation during spoilage of several fresh water, estuarine. and marine fish from South India. Histamine was absent from fresh meat in all species. After 72 hours of storage at 30”C, significant quantities of histamine were formed, particularly in the marine species. However, levels were not greater than 15 mg/100 gm tissue which is considerably less than levels generally associated with histamine toxicity. Ferencik and Havelka ( 1 962), having examined a large number of samples of tuna muscle of different types held at varying temperaturcs. concluded that the histamine level was a good indicator of freshness.

Hillig (1956) has reported the results of a very detailed study in which several species of tuna were analyzed as they were allowed to reach different stages of decomposition and were subsequently precooked and canned in the usual man- ner. Different sections of individual fish were evaluated as were fish from different boats. A number of chemical analyses were employed, together with organoleptic evaluation. It was found that canned tuna prepared from good fish normally contain a small amount of acetic acid. and that as decomposition progresses there is an increase in acetic acid content as well as that of formic acid. After further decomposition, propionic and butyric acids may appear. Hillig suggests that individual volatile acids in canned tuna are a good index of the stage of decomposition of the corresponding raw material. Succinic acid and histamine were also formed during decomposition, with histamine reaching levels of well over 1000 mgll00 gm, dry weight basis. Some of the indices were partially lost during canning but the losses were considered not to be sufficient to enable a decomposed fish to become passable when canned.

In another in-depth study. Takagi ef L I / . (1969) examined the amounts of histidine and histamine in 21 species of aquatic animals during spoilage. Their findings were in agreement with those of others already summarized here in that more histamine was produced in the “red muscle” fishes such as mackerel species than in ”white muscle” fishes such as rockfish. They also found little or no histamine produced in several molluscs and crustaceans during storage. Within the same species of fish, more histidine was found in white than in red muscle, and the resulting histamine formation followed this pattern with regard to muscle type, i.e., more histamine in white muscle. Of interest is their observation that while the degree of histamine formation is governed by histidine con.- tent. it is not proportional to the loss of histidine. Recently, Cantoni o r a/ .

I38 SALLY HUDSON ARNOLD AND W. DUANE BROWN

(19764 compared histamine production with that of volatile acids and volatile bases in tuna stored at 18”-20°C. They report that toxic levels of histamine were noted after 4 days; this level was apparently defined as greater than 100 mg histamine per 100-gm fish. During the same 4-day period the development of high levels of volatile acids and bases was also noted.

Thus, there is considerable evidence associating several of the objective measurements of fish deterioration with histamine levels. Clearly, however, no objective measurement, short of determination of histamine itself, emerges as an effective indicator. As discussed in the section on analytical determination of histamine levels, efforts are now being made to reduce the measurement of histamine to a rapid, routine analysis.

In the tuna industry, routine quality control consists of inspection of the raw fish. Organoleptic grade classification has been employed. Such grades are based on evaluation of physical characteristics, including appearance of gills, eyes, and skin, smell, and degree of physical damage to the tuna (Lassen, 1965). Such inspection would not, of course, reveal anything about histamine levels. All commercially canned tuna is precooked. Following the precook, skin and bones are removed and the loins are cleaned. At this point a phenomenon known as “honeycombing” may be observed. This condition consists of areas of pitted, spongy-looking muscle tissue (Finch, 1963). It is apparently due to gas production accompanying microbial growth. and is the basis for rejecting for human consumption the fish in which it is found. While this problem is widely known, if infrequently encountered in industry, it has been the object of little research, or at least little published research. Otsu (1957) reported that honeycombing developed in Hawaiian skipjack held without refrigeration, independent of sexual maturity or size of fish. He noted that honeycombing resulted from delayed rcfrigeration in experiments performed at different times of the year, although the rate of honeycombing was more rapid at higher seawater temperatures. He suggested that since the months of highest water temperatures in Hawaii coincide with the peak occurrence of honeycombing noted by fishermen, there might be a close relationship between water temperature and honeycombing. In a study by Williams (1954) in which several species of fish, including yellowfin and skipjack tuna, were allowed to reach varying stages of decomposition, much higher histamine values were found in honeycombed fish, regardless of species, than in similar fish without honeycomb. Merson et al. (l974), reporting on the incident of scombroid fish poisoning traced to commercially canned tuna in 1974, stated that fish from incriminated lots showed evidence of honeycombing. This is somewhat perplexing in that, under ordinary circumstances, tuna showing signs of honeycombing would have been rejected, as indicated previously.

Researchers in the field, as well as industry representatives, unanimously agree that canned tuna with high levels of histamine imparts a “peppery” feel to the mouth when chewed. However tasting per se on a routine basis does not seetn


a feasible means of quality assurance. At present, individual lots of tuna are analyzed for histamine content. This is, at best, a tedious procedure. As mentioned earlier, the relationship between detectable olfactory changes and histamine content is not always evident (Fucker ef al., 1974).

Thus, there remains a need for an efficient objective measure of decomposition or a more rapid histamine analysis. Kimata ( I96 I ) has suggested that freshness of fish kept at temperatures as high as 35°C may be judged by determining the content of ammonia, since production of histamine under these circumstances is negligible as compared to that of ammonia. However, the use of ammonia levels as a freshness indicator becomes unreliable when fish are kept at room temperatures (20°C). He includes data showing that production of histamine in certain fish predominates over ammonia production in the range of temperature from 6 to 20°C. This is unfortunate since electrodes specific for ammonia are available and conceivably could be used on a routine basis.

In that connection, in a recent report by Chang et al. (1976) there appears the description of the use of a trimethylamine-specific electrode for fish quality control. The electrode can be used for measurement of trimethylamine in aqueous solutions as well as in homogenates of fish muscle. It certainly offers a much simpler means of analysis than other methods currently used for determining trimethylamine.

Some readers may be familiar with the Torrymeter, a device developed by research at the Torry Research Station in Aberdeen, Scotland. It is a compact portable meter which measures changes in dielectric properties of raw fish muscle and skin which occur as freshness is lost. The sensing head of the meter, containing the electrodes, is pressed against the fish skin, and a reading appears. The actual numbers read decrease with deterioration in quality. The device is simple to use and has practical application. Unfortunately, there seems little potential for comparing quality measured by the Torrymeter with histamine production, inasmuch as it cannot be used on fish that have been frozen (as are most tuna, for example) and it is said to be unreliable on fatty or oily fish such as tuna and mackerel (Davis, 1976).

V. UNRESOLVED PROBLEMS

A. IS SCOMBROID TOXICITY DUE TO HISTAMINE?

In our considered judgement, the answer to this question is no. At least it seems extremely unlikely that histamine, acting alone, is the sole factor responsible for scombroid poisoning. A number of reports indicate that histamine taken orally by human subjects is not toxic. Douglas (1970) states “Very large amounts of histamine can be given orally, however, without causing ef-


fects. . . .” He attributes this to the conversion of histamine to inactive N-acetylhistamine by intestinal bacteria. Granerus ( 1968) gave human subjects up to 67.5 mg histamine orally and reported that “The subjects did not get any subjective or objective symptoms which might have been caused by histamine.”

A particularly convincing report is that of Weiss et al. (1932). They fed normal volunteer subjects gradually increasing amounts of histamine phosphate from 200 to 500 mg (500 mg histamine phosphate is equivalent to about 180 mg of histamine base). They noted no subjective or objective changes. Pulse rate and arterial blood pressure were unaltered. Readers are cautioned to note carefully the form in which histamine is given in such studies. In this case the phosphate salt was used, as noted. The dihydrochloride salt is sometimes used; 100 mg of histamine dihydrochloride is equivalent to about 60 mg histamine base.

Hardy and Smith ( I 976) point out that Hughes ( 1 959) found higher values of histamine during postmortem storage of herring than they found in mackerel. Of interest is their comment “yet in this country only the latter is alleged to cause illness.” Typical values in the two reports were about 22 vs 2.6 at 80 hours storage. These are both below histamine levels usually regarded as toxic.

Although unreported to date, it is also known that several individuals currently investigating the histamine problem have themselves consumed sizable quantities of pure histamine as well as good tuna “spiked” with histamine (up to 100 mg) with no apparent effect.

There are few papers in which a response to oral histamine has been noted. In one such report (Sjaastad, 1966), subjects given doses of histamine of 36 mg or more experienced nausea, belching, heartburn, borborygmia, and diarrhea. We are at a loss to explain this apparent contradiction except to note that these symptoms are generally inconsistent with those normally associated with so- called histamine toxicity.

Thus, it is difficult to conclude that histamine per se is the causative agent. This is not to say by any means that histamine is not involved. It seems beyond question that there is a direct correlation between high levels of histamine in fish and the resulting scombroid toxicity when such fish are consumed by humans. It is equally obvious that histamine alone is not responsible and, therefore, there must be some accompanying synergistic substance(s) or potentiating condition.

With regard to the role of histamine, it should be noted that much of the histamine normally stored in tissues is found in mast cells or in basophils in the blood. In these cells, histamine is stored as a complex with heparin in secretory granules. While the turnover rate is slow, histamine may be caused to be released by a variety of factors, including allergy, various drugs, physical or chemical insult. Such release could account for some, if not all, of the symptoms associated with scombroid toxicity. For more detailed information concerning the mode of action of histamine see Douglas (1975) and Beaven (1976a,b).

HISTAMINE (‘0 TOXICITY FROM FISH PRODUCTS 141

B. IMPROVED ANALYTICAL PROCEDURES

Recent research in this area, including some not yct completed, has been quite responsive to the needs. Several new methods have been developed as mentioned in Section 111. If these methods prove to be as promising a s they appear. this may no longer be highly critical rescarch. A triore rapid “official” method is being developed (see Section 1LI.C).

On the other hand, the need for an improved bioassay is critical indeed. It is scarcely feasible to employ human subjects. but other animals tested to date have responded at best in a highly variable fashion. Geiger (1955) fed samples of tuna which had been purposely spoiled. and which had levels of histamine ranging from 190 to 210 mg per 100 grn, to dogs. cats, rats, and mice, and by stomach tube to guinea pigs. No symptoms of poisoning were observed. Work in progress confirms the lack of toxicity of tuna known to cause illness in humans when fed to dogs and cats. The same worker has noted growth impairment in Japanese quail fed toxic tuna or histamine (Blonz, 1976). I t has been reported earlier that toxic tuna meal or pure histamine fed to chicks resulted in growth inhibition (Shifrine rt d., 1959). The fact that histamine alone inhibits growth may miti- gate against the use of quail or chicks as a bioassay inasmuch, as previously indicated. histamine alone taken orally does not appear to be the sole toxic factor in scombroid poisoning. This same reservation applies to use of the guinea pig ileum assay inasmuch as that muscle contracts in response to pure histamine. It will be of interest to see if the work cited earlier (Blonz. 1976) with Daphnia, a fresh water crustacean. yields a new bioassay for the toxic substance(s). Blonz is also presently testing pigs as experimental animals. In a preliminary trial. one pig weighing about 73 kg was fed 900 gin of toxic canned tuna. The animal was observed for several hours. but no signs of distress were noted.

C . THE ANSERINE AND CARNOSINE QUESTION

Carnosine (P-alanylhistidine), and anserine (N-P-alanyl- 1 -methyl-histidine) are present in muscle tissue of a number of animals, including tuna and other fish. Structures are shown in Fig. I , together with those of histidine and histamine. Analyses for levels of these two dipeptides in a variety of fish have been reported by Lukton and Olcott (1958). Yellowfin tuna light meat contained small amounts of carnosine, averaging about 1 pmole/grn. with some samples showing


COOH H H H2N-CH-CH2fi HpN-CH2-CHzfi

Histidine Histamine

0 7OOH 7H3 YOOH H II

(1 f] H2N-CH2-CH2-C-NH-CH-CH2 H2N-CH2-CH2-C-NH-CH-CH2 51

Carnosine Anse r i ne (N -8- A Ian y I hist id i ne 1 (N-8-Alanyl-1 -methyl histidine)

FIG. 1 . Structures of histidine, histamine, carnosine, and anserine.

zero levels. Anserine, on the other hand, was present in significant amounts, averaging about 29 pmole/gm. The significance of the potential contribution of anserine to histamine formation (if this, in fact, can occur) can be seen from the fact that histidine levels in these same loins averaged about 39 pmole/gm. In light meat of albacore tuna, typical values were: carnosine, 3, anserine 25, and histidine 45 pmolelgm, respectively. In skipjack tuna light meat, corresponding values were: carnosine, 4, anserine, 16, and histidine, 53 pmolelgm, respectively. Assays were also made for L-methylhistidine, but detectable levels were not found in any of the tuna samples.

It was shown many years ago that E . coli can hydrolyze carnosine and decarboxylate the histidine liberated (Nash, 1952). Under the conditions employed by Nash, the pH range was critical, the organisms accomplishing the decarboxylation at pH 4.0. Hanson and Smith (1949) had earlier described a carnosinase found in swine kidney. Other work has been done with these dipeptides, but to our knowledge none has been directed to the question of the possible contribution of either to the production of histaminelike substances in scombroid fish. Two questions suggest themselves, one being whether enzymatic systems present in fish or in microflora commonly associated with fish spoilage can hydrolyze these peptides and decarboxylate histidine to produce histamine. A second is whether decarboxylated carnosine and anserine have any histaminelike activity.

D. POSSIBLE SYNERGISTS OR POTENTIATORS

If, as seems clear, histamine itself is not toxic when taken orally, but, also, if histamine is found in large amounts in fish and other foods known to cause illness when consumed, then it is evident that there must be synergistic or potentiating substances or conditions involved. A number have been suggested, as outlined below. However none has been clearly implicated, and the evidence available at

HISTAMINE (’?) TOXICITY FROM FISH PRODUCTS 143

presetit, when taken as a whole, is largely negative. Thus, there is a pronounced need for additional research in this important area.

I . Other Amines

It has been suggested by a number of investigators that other amines, including certain diamines, may work synergistically with histamine to produce toxicity. Miyaki and Hayashi (1954) reported finding a “factor” in a dried fish product which worked cooperatively with histamine to cause food poisoning. Hayashi ( 1954) subsequently reported that trimethylamine oxide, trimethylamine, agmatine (decarboxylated arginine), and choline worked synergistically with histamine in causing contraction of guinea pig uterus. However, Kawabata et al. (1 9S6a) reported that the addition of either trimethylamine or trimethylamine oxide was without effect on the action of histamine on guinea pig uterus and concluded that they could not be involved as cooperating factors in “allergy-like food poisoning.”

Aipo et al. (1967) inoculated heat-sterilized Pacific saury (Cololubis saira) with Proteus morganii and, following incubation for 48 hours, chromatographed alcoholic extracts of the inoculated material. While they found that more than 90% of the histidine in controls had been converted to histamine during the incubation, they could not detect the presence of agmatine, cadaverine, methylhistamine, or tyramine. Furthermore, of the eleven fractions they isolated, only the one containing histamine resulted in any activity in guinea pig intestine.

There is some evidence that certain amines may influence the absorption of histamine. This material is covered in Section V,D,2, immediately following.

2 . Alterations in Absorption

Since large amounts (180 mg) of histamine can be taken orally by man without effect while microgram quantities in the blood may cause systemic effects, there must be some inactivation before histamine reaches general circulation. Weiss et al. ( I 932) suggested that either histamine is inactivated in the intestines before it enters the portal circulation or is destroyed by the liver before it enters from the portal to general circulation. They favored the former conclusion. More recently, Douglas (1970) reported that histamine given orally is converted by intestinal bacteria, particularly Escherichia coli, to inactive N-acetylhistamine, and that any free histamine remaining is inactivated when it traverses the intestinal wall or passes through the liver. Sjaastad ( 1 966) had shown previously thal almost 80% of N-acetylhistamine administered orally to humans was excreted as such in the urine. Thus, the acetylated derivative apparently is absorbed with no difficulty.

There is, however, some evidence that other materials, if present with his-


tamine, may alter its absorption. Parrot and Nicot (1 965) reported that histamine is normally fixed by much in the gut, but that certain diamines, such as putrescine, may overcome the fixation. In their study, the administration of putrescine before that of histamine enhanced the toxicity of the latter. Ienistea (1973), in reviewing the work of Parrot and Nicot just cited and that of earlier workers, concluded that diamines may enhance the toxic action of histamine by facilitating its passage through the intestinal barrier. He further suggested that the association of several diamines, such as putrescine, cadaverine, and spermine, with histamine may exert deleterious effects on animals.

Geiger ( 1 955) noting the work of others and his own earlier findings suggested that alterations of conditions of the intestinal tract might cause histamine to be absorbed at an increased rate such that detoxification could not keep up with the entry of histamine into the circulation. Two such conditions he postulated were the consumption of highly seasoned hot dishes prepared from spoiled fish or the simultaneous consumption of alcoholic beverages.

Mitchell and Code (1954) reported that, when healthy adult humans were fed 60 mg of histamine, large amounts of conjugated histamine were found in the urine during the third hour after administration. None of the ingested histamine appeared in the urine as free histamine. However, they did note that when histamine was added to a meal (consisting of bread, butter, and milk) that there was an increase in excretion of the conjugate comparable to that following consumption of histamine alone, but there was also a sharp increase in the output of free histamine in the urine starting about 2 hours after the meal containing histamine. The authors suggested the hypothesis that the absorption of free histamine is increased in the presence of the meal. However no direct evidence on this point was presented.

It was earlier noted herein that Weisset al. (1932) had fed up to the equivalent of 180 mg histamine base to human subjects without noticeable effect. The same investigators showed that the minimal single intravenous injection of histamine that would elicit changes in facial blood vessels and cardiac rate was 0.007 mg of histamine base. Thus, whatever the mechanism preventing intestinal absorption and/or subsequent entry into the general blood circulation, it must be extraordi- narily efficient.

3 . Histaminase

Widmann ( I 950) obtained a patent on the invention of a method for increasing the absorption of histamine by supressing its inactivation by histaminase. Mate- rials he suggested using for histaminase inactivation included histidine. pyridoxine, cysteine, ascorbic acid, ethylenediamine and its hydrochloride salts, and tetramethylenediamine and its hydrochloride salts. He indicated obtaining successful clinical results with these materials, based on showing a vasodepres-


sor effect on hypertensive individuals. He was defining histaminase as a “diaminooxidase.” In this connection. Douglas (1970) noted that, while the most important pathway of histamine metabolism involves ring N-methylation by histamine-N-methyltransferase, there is another pathway in which histamine undergoes oxidative deamination by enzymes known as histaminase or diamine oxidase. For additional coverage of histaminase, see Section 1I.H.

4 . Monoamine Oxidase Inhibitors

There is at present widespread use of monoamine oxidase inhibitors as an- tidepressant drugs. There have been frequent reports of problems (hypertensive disturbances, headache, palpitation. and flushing) encountered by patients using such drugs following consumption of food products known to contain certain amines such as tyramine (decarboxylated tyrosine). A brief review of this problem is available (Anon., 1965). Sen ( 1 969) surveyed a variety of food products for their tyramine levels. Significant levels of tyramine were found in a variety of cheeses and yeast extracts, with lesser amounts being found in meat extracts and salted fish. More recently, Voigt ef al. (1974) and Rice rt al. (1975) have reported results of analyses of a number of cheeses and meat products, respect- fully. for content of histamine and tyramine. Histamine concentrations in cheese ranged from nondetectable amounts to 2.6 mglgm in a sample of Sap-Sago. Histamine was detected in semi-dry sausage products and country-cured hams, but amounts were very low, in the order of 0.002 to 0.003 mg/gm.

These and other reports have triggered speculation that monoamine oxidase inhibitors might potentiate the effects of ingested histamine. While this cannot be ruled out on the basis of evidence at hand, it does seem unlikely to be responsible in a majority of the cases of scombroid toxicity. For example, Boyer et a/ . ( 1 956) reported one incidence in which the proportion of victims becoming ill following consumption of tuna with high histamine levels was 400 out of 2500 people eating the product. It is clearly unlikely that all 400 individuals were taking monoamine oxidase inhibitors.

5 . Bacterial Endotoxins

Bacterial endotoxins are known to be widespread. These complex lipopolysac- charide materials are produced primarily by gram-negative bacteria and are known to be relatively heat stable. These factors have led t o speculation that such endotoxins might act synergistically with histamine in scombroid fish which had been subjected to significant microbiological spoilage. It should also be noted that endotoxin is known to be capable of inducing histamine release in animals (sometimes called endotoxin shock) similar to that seen in anaphylaxis. I t also causes hypersensitivity to histamine in some animals. However, these

I46 SALLY HUDSON ARNOLD AND W . DUANE BROWN

phenomena are observed when endotoxin is given intravenously, and when this is followed by intraperitoneal injection of histamine. No effort will be made here to review this literature. The following citations offer typical experimental findings (Hinshaw et al., 1961; Kuratsuka and Homma, 1975; Kuratsuka et al., 1975).

We have found no reports in which endotoxin and histamine were fed to animals to determine their combined toxicity. In preliminary experiments, Baronowski (1976) has found only extremely low levels of endotoxin in both good tuna and in that known to have caused illness when fed to humans. These findings need to be confirmed and extended to rule out endotoxin involvement. For detailed coverage of endotoxin see Weinbaum et al. ( I 97 1) and Kadis ef ul. ( I 97 1).

6. Conclusions

Of the suggested routes by which synergistic materials may act, or by which histamine toxicity may be potentiated, those deserving of additional study seem to be (1) the effects of other amines, such as putrescine, and (2) various factors that may influence histamine absorption. Since such widely disparate food products as canned tuna fish and cheese (Douglas et al., 1967) may induce histamine toxicity, it seems not unreasonable to assume that the additional factor(s) may be microbial in origin. Clearly, there may well be other contributory substances or conditions which are yet to receive consideration. Identification of synergists and/or potentiators may well result in solution of the problem of “histamine” toxicity.

E. ALLOWABLE LEVELS OF HISTAMINE IN FISH

At the present time, the U.S. Food and Drug Administration is known to be considering establishing maximum allowable levels of histamine in canned tuna fish. Since histidine is a normal constituent of tuna muscle, and since there will always be some microbial flora associated with fish regardless of the care with which they are handled, it is inevitable that some histamine will be produced. Thus, there arises the question of what levels may be accepted in good quality fish. At the time of this writing, no number has yet been officially proposed. However, the maximum allowable level most frequently discussed unofficially is around 10 mg histamine per 1 0 gm fish. It is of interest to note that Geiger (1944a) long ago said that “We do not feel ready to set standards for the freshness of the fish in terms of the histamine content, but on the basis of our experimental data we assume that fish containing more than 10 mg ‘histaminelike’ substances per 100 gm tissue should not be regarded as ‘fresh’.” It may well be that Dr. Geiger’s educated guess will prove to be prophetic.

HISTAMINE (? j TOXICITY FROM FISH PRODUCTS 147

ACKNOWLEDGMENTS

Much of the work mentioned herein as being in progress is supported by the Tuna Research Foundation and NOAA Office of Sea Grant. Department of Commerce. under Grant No. 04-6-158- 44021. Ms. Arnold is a Sea Grant Trainee.

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Aiso. K . , lida, H.. Nakayama, I . . and Nakdno. K . 1958a. Histamine poisoning caused by certain marine fish products and the specific etiological agents. Chiba Daigaku Fuhni Kenkyusho Nokoku 11, 1-6.

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[advances in food research] volume 24 || histamine (?) toxicity from fish products

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