abnormal trace metals in man-vanadium* · abnormal trace metals in man-vanadium* henry a....

J. chron. Dis. 1963, Vol. 16, pp. 1047-1071. Pergamon Press Ltd. Printed in Great Britain

ABNORMAL TRACE METALS IN MAN-VANADIUM*

HENRY A. SCHROEDER, M.D.?, JOSEPH J. BALASSA, Ph.D. and ISABEL H. TIPTON, Ph.D. Department of Physiology, Dartmouth Medical School, Hanover, New Hampshire; Brattleboro Retreat, Brattleboro, Vermont; Department of Physics, University of Tennessee, Knoxville, Tennessee

(Received 5 February 1963)

VANADIUM was discovered in 1801 by DEL RIO who called it erythronium. Later he decided that it was a form of chromium. Not until 1831 was it finally identified by SEFSTR~M who namea it after Vanaciis, the race of Freia, the Norse goddess of beauty [l]. Such beauty as it may have is chemical, lying in its excellent catalytic properties and physical, in the color of its compounds, but not particularly in the green tongues it causes in exposed workers [2].

During the age of metallotherapy (perhaps now due for a renaissance) compounds of vanadium were given therapeutically for a diverse variety of disorders; as antiseptic, spirochetocide, anti-tuberculous and anti-anemic agents; to boost resistance to infection and as tonics to improve appetite, nutrition and general health [I]. Obviously vanadium was not very toxic, for the doses employed were often large, 150 mg of the sodium tartrate intramuscularly and l-8 mg of the metavanadate by mouth [3]. Its use was largely given up two decades or more ago.

To the BERTRANDS, p&e et jils, is largely owed interest in the possible biological activities of vanadium. Since 1903 some 18 of their publications have appeared, one on work done under the severe conditions of the German Occupation of Paris [4]. Partly because of their continuing investigations, vanadium as a trace metal has occupied a position of theoretical importance to plant and animal life for over half a century. Further interest was aroused when its activity on two currently popular substances was demonstrated. CURRAN [5] showed that vanadium suppressed the hepatic synthesis of cholesterol and offered evidence that it might inhibit experimental atherosclerosis [6]. PERRY et al. [7] found enhancement of activity of monoamine oxidase, an enzyme which long ago was shown to be acutely anti-hypertensive in rats and dogs [81 and which inactivated angiotensin [8-101.

The purpose of this report, the sixth of a series, is to examine vanadium from its biogeochemical aspects in order to decide whether or not it behaves as an essential trace metal in man. To do this, it was necessary to evaluate distributions, mammalian exposures, biological activities and clinical experiments, and to estimate exchanges and balances. Some of our findings were surprising.

METHODS Most methods for the estimation of vanadium in biological material leave something

to be desired. Emission spectrographic analyses, of which many have been reported in

*Supported by grants in aid from the National Heart Institute, United States Public Health Services (H-5076), the Vermont Heart Association and Ciba Pharmaceutical Products, Inc.

tRequests for reprints should be sent to 75 Linden Street, Brattleboro, Vermont.

1047

1048 HENRY A. SCHROEDER, JOSEPH J. BALASA and ISABEL H. TIPTON

the past, included vanadium present in carbon electrodes; graphite electrodes free of vanadium are now available. Chemical analyses using cupferron were uncertain as many metals were chelated thereby. The method of SANDELL [l 1 ] using the hydroxyquinolate of vanadium, was moderately sensitive and reproducible, but in our experience indicators controlling the hydrogen ion concentration interfered with the absorption spectrum of the hydroxyquinolate in chloroform. SANDELL’S method was modified, substituting n-amyl alcohol (which gives a red color) for chloroform, controlling pH with an electrode or with test paper and reading optical density at 500mp. Ironwasremovedby the usual ammoniacal precipitation in the presence of ammonium chloride.

The method was found to be reasonably accurate, mean recovery of known quantities added to 27 samples being 94.5 per cent with a standard deviation of + 2.58 per cent. In 21 cases recovery was 96 per cent. Components of a rat and mouse diet, rye, powdered skim milk and corn oil, were analyzed separately and compared to analysis of the finished diet; the difference was 5.5 per cent. Sensitivity of the method was good, the limit of detection being < 0.5 pg per sample of 10-100 g, or 0.05-005 pg/g wet weight.

Vegetable foods were obtained from a home-grown garden, the garden of the Brattleboro Retreat and local chain stores, from which meats and fish were also pur- chased. Wild animals were trapped, shot or accidentally killed on the highway. Grains were supplied by local feed stores, oils and fats from commercial sources.

Samples were dried and ashed in silica crucibles in a muffle furnace at 400°C after careful washing in deionized water. Fluids were evaporated to dryness before ashing. Oils and fats were saponified in order to avoid loss while ashing. Most human tissues were analyzed by emission spectrography as previously reported [12], being carefully dissected free of fat.* Specimens from Africa were obtained by H. M. PERRY, Jr., from the United States by M. J. COOK and from the remainder of the world by one of us (H.A.S.). Cholesterol in serum was measured by the method of ABELL et al. [13].

Vanadium in human tissues RESULTS

In American organs and muscle tissue vanadium has been generally detected by the spectrographic method only in lung (51.7 per cent) and intestine (15.7 per cent). It was found in no samples of heart, aorta, brain, kidney, muscle, ovary or testes. Most positive samples showed 0.01 pg or less per g of tissue. It was present in one adrenal, prostate, spleen, thyroid and uterus; in two of 139 specimens of pancreas, three of 22 skins and five of 148 livers. In 630 samples from the gastrointestinal tract, it was more or less localized to ileum (37 .O per cent), cecum (45.1 per cent), sigmoid colon (15.8 per cent) and rectum (26.2 per cent), being infrequently found in esophagus (7.6 per cent), stomach (2.3 per cent), duodenum (7.5 per cent) and jejunum (3.9 per cent) [14]. Fat and serum were not analyzed by this method.

There were some geographical variations in the frequency of occurrence of pulmonary vanadium in the United States (Table 1) and in various regions of the world (Table 2). In fact, vanadium was retained by a number of kidneys and livers from the *Values for human tissues are reported in terms of wet weight or ash, depending upon the original source. All other values are given in terms of wet weight, for these are the concentrations ingested, or present, and possibly biologically active. The authors will supply dry and ash weights upon request. When 0.0 is shown in the tables, it indicates a value less than 0.05 pg/g.

Abnormal Trace Metals in Man-Vanadium 1049

TABLE 1. VANADIUM IN LUNG AND AIR, UNITED STATES VALUES

City Number Number % M~~II~s.E.M.* Range Air?

of found found (kg/g wet) &g/g wet) samples Found &g/m?

(%)

San Francisco, Calif. 27 22 81 0.06~0.009 <0.02-0.18 - - Baltimore, Md. 24 17 71 0.06+0.013 <0.01-0.24 100 1.000 Seattle-Tacoma, Wash. 19 13 68 0.10f0.03 <0.01-0.48 93.9 0.087 Denver, Colo. 24 16 67 0.1010.03 <0.01-0.68 36 0.250 Richmond, Va. 25 12 48 0.13+0.05 <0.01-0.95 - - Dallas, Tex. 28 11 39 0.02*0.005 10.014.12 20 0.012 Miami, Fla. 26 6 23 0.0110.004 <0.01-0.06 - -

*Mean values were calculated from all samples, assuming that those with undetectable amounts contained one-half the value of the limit of detection, i.e., 0.005 pg/g.

tconcentrations in air from Air Pollution Measurements of the National Air Sampling Network, 1957-1961. U.S. Dept. Health, Education and Welfare, 1962.

TABLE 2. VANADIUM IN KIDNEY, LIVER AND LUNG. GEOGRAPHICAL AREAS

Mean* Range Median Area No. of %

samples found (p.p.m. ash)

Kidney United States Switzerland Africa Middle East Far East

Liver United States Switzerland Africa Middle East Far East

161 9

44 43 57

163 4 9 0

40 0 44 18 54 7

0 0 2

23 7

- - - - - - 0.5 <l <l 1.3 <l-3.6 il 0.6 <I <1

il 11-4.8 <I - - - - - -

(1 <l-5.7 il il <l-4.8 <l

Lung United States 159 58 12 i l-110 <l Switzerland 7 100 3.2 cl-11 2.4 Africa 39 59 4.0 < l-22 3.0 Middle East 45 76 10 < l-50 6.0 Far East 57 60 4.0 < l-29 <l

*Mean values of positive samples.

Middle East where its median value in lung was also highest. These data suggest that vanadium can be absorbed from the environment under certain (unknown) conditions, and that its presence in man is not always confined to organs exposed to air and food.

Accumulation in human tissues with age was calculated on the limited number of positive samples. Vanadium was detected in only one infant less than 6 weeks old, in lung, and in only one child under 10 years of age. The data are indefinite as to positive increases with age, either in lung or intestine and no clear tendency for steady accumulation under natural exposures appeared, unlike the case of cadmium, lead and titanium [15-171.

1050 HENRY A. SCHROEDBR, JOSEPH J. BALA~~A and ISABEL H. TPTON

A 75-year-old patient was given 4.5 mg vanadium orally as diammonium oxytartarovanadate daily for 12 months. Postmortem analyses revealed none detectable in muscle, 3.38 p.p.m. (wet weight) in liver and 5.83 p.p.m. in subcutaneous fat (Table 3), indicating that under unusual exposures vanadium can be retained by liver in amounts approximately 50 times the largest found from natural exposures (0.07 p.p.m.). Samples from a patient not unduly exposed also contained considerable vanadium by our method.

Paired rank correlations of vanadium and 15 other trace metals in 29 tissues were determined by digital computer. Choosing r > 0.50 and P=O. 001, vanadium was associated with manganese in lung. With r < 0.50, it was found with chromium, copper and lead in the gastrointestinal tract and with boron, nickel, tin, titanium and chromium in lung. At a lower level of significance (P=O. 005 and r < 0.50) vanadium was found associated with manganese, molybdenum and iron in various portions of intestine. Apparently the affinity of vanadium for these two types of tissue was shared by chromium and manganese, while most other organs failed to accumulate it.

TABLE 3. VANADIUM IN ANIMAL TISSUES

Animal Tissue @g/g wet wQ Remarks

Rat, Long Evans 3

Mouse, white 9

Beaver d

Woodchuck ?

mrr3 Deer $?

Rabbit, domestic, fat, white d

Horse Chicken Human, 9, age 3 Human, ?, age 65 Human, 9, age 40 Human, d, age 60

Human, d, age 76

Kidney 0.0 Liver 0.39 Lung 0.0 Whole, young 0.0 Liver 0.0 Mammary tumor 1.91 Mammary tumor 1.54 Kidney 0.0 Heart 0.0 Kidney 1.08 Liver 0.0 Lung 0.0 Heart 1.11 Spleen 1.16 Kidney 2.07 Liver 0.07 Heart 0.13 Aorta 0.0 Hoof 2.55

Kidney 0.59 Liver 0.94 Lung 0.0 Heart 3.40 Blood 0.0 Heart 0.0 Hair, blond 0.0 Hair, red 2.71 Hair, brown 2.59 Muscle, fatty 1.19 Muscle, extracted 0.0 Liver 1.88 Muscle 0.0 Liver 3.38

On diet containing 3.2 p.p.m. V

On diet containing 3.2 p.p.m, V

Cf. Tables 1 and 2

See gelatin, Table 4

Diet high in V

Cf. Tables 1 and 2

‘Normal’ V intake Fat removed by hexane

Given 4.5 mg V daily for 12 months

Note: Compare analyses of domestic animal tissues, Table 4, for beef, pork and lamb, kidney, liver and muscle.

Abnormal Trace Metals in Man- -Vanadium 1051

Vanadium in animal organs Results of a pilot survey of some wild and domestic animal organs are given in

Table 3. Laboratory rats ingesting 3.2 p.p.m. in food (Table 4) accumulated no detectable vanadium in kidney, but some appeared in liver. A newborn animal was free of this metal. Two mouse mammary tumors showed much vanadium. Unlike the case of man, none was detected in the three lungs analyzed. No apparent regularity of occurrence was evident from this study, either by species or by organ, although many organs of wild animals contained much larger quantities than were found in the ,same ones of human beings. On the other hand, vanadium was conspicuously absent from muscle, liver and kidney of domestic animals (Table 4). An apparent affinity for keratinaceous tissue appeared in the results for hooves, hair and gelatin.

Vanadium in foods

Analyses of some 100 common foods revealed irregular but rather high exposures of human beings to vanadium (Table 4). Institutionalized patients ingested 1.16 mg during a sample day’s diet, and calculations indicated that even larger amounts were not unusual (2-3 mg). No appreciable vanadium was present in butter, cheese, fish, meats, wheat, white bread and millet. Large amounts (> 1 p.p.m.) were contained in skim milk powder, two sea foods, gelatin, oats, rye, buckwheat, a breakfast food, and many vegetables, leafy, root and leguminous, most of them grown in soil with 1.36 p.p.m. soluble vanadium. White turnips, onions, garlic, lima beans, 4 fruits and 2 of 6 nuts were deficient.

That great differences in intake could occur is shown by the institutional diet, where concentration in the breakfast was 0.02 p.p.m. and in the supper 1.03 p.p.m. Analyses of the foods accounted for these variations, the main caloric intake of the breakfast being vanadium-poor oatmeal and wheat flour products, while the supper contained green vegetables, macaroni and gelatin.

To ascertain the uptake of vanadium by grains from long-uncultivated soil, wheat rye, buckwheat and oats were grown on unfertilized land reclaimed from an old overgrown pasture in a forest, with 0.57 p.p.m. available in the soil. Seed wheat without vanadium produced kernels containing it. Oats and seed rye with vanadium lost it when mature. Buckwheat was little affected, seed and mature grain having similar amounts.

Vanadium in fats and oils

Vanadium has a natural affinity for biogenic fats and oils. Large amounts were found in soybean, corn, olive, linseed and peanut oils; castor oil was the only vegetable oil deficient (Table 5). This phenomenon was hitherto unknown. Soybean oil had over 40 p.p,m. Nine hundred calories of these oils would supply an intake of l-4.4 mg of vanadium. Extraction of rye flour with benzene showed that all of the vanadium was in the fatty portion.

The affinity of vanadium for animal fats was also clear. Pork, deer and chicken fats had large amounts, while none was found in meat. No vanadium was detected in lard, butter or cod liver oil. Sperm oil, however, containing about 42 per cent of unsaturated fatty acids and alcohols [18] had much vanadium. The patient given vanadium orally had accumulated it in subcutaneous fat in fairly large concentrations, while one unexposed had lesser amounts.

1052 HENRY A. SCHROEDER, JOSEPH J. BALASSA and ISABEL H. TIPTON

TABLE 4. VANADIUM IN FOODS

Caloric values calculated from MCCANCE, R. A. and WIDDOWSON, E. M.: The Chemical Com- oosition ofFoo&, Chemical Publ. Co., Brooklyn, New York, 1947.

Item &g/g &g/l00 Item @g/g (cIg/lOO wet wt) calories) wet wt) calories)

MILK AND DAIRY PRODUCTS Milk, dried skim Milk, whole, local Butter, salted, local Butter, unsalted, local Cheese, Swiss Cheese, spread Egg yolk Egg white

FISH AND MEAT Scallops Oysters Lobster : claw meat Lobster: digestive gland Halibut Cod, dried Beef, chuck Beef, kidney Pork chop Pork liver Pork kidney Lamb chop Lamb liver Chicken breast Gelatin

VEGETABLES Potato* Beets* Carrots* Radishes* Radish leaves* Turnips, yellow* Turnip greens* Turnips, white Turnip greens Onions* Garlic Peas, green* Pea plants* Peas, split green, dried Peas, green, home grown Beans, lima* Bean plants* Beans, string* Beans. Naw. dried Lentils, dried Squash, Hubbard Lettuce, iceberg Swiss Chard* Cabbage Mushrooms

6.52 200 0.01 2 0.0 - 0.0 - 0.0 - 0.0 - 0.68 17 0.37 100

2.1 0.11 5.1 2.25 0.0 0.96 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.14

12.59

200

4Z - - 30 - - - - - -

7 -

485

1.49 0.88 0.99 3.02 2.53 0.73 1.47 0.0 0.02 0.06 0.0 3.27 2.13 0.65 0.46 0.0 0.55 0.87 5.04 6.00 1.23 1.08 1.04 1.75 0.0

171 200 430

2013 -

405 1336

- 18 25 -

511

u 72 - -

1243 195 200 820 982 945 875

-

Mean 1.47 523

‘RUITS Tomato 0.0 - Blackberries, wild 0.0 - Avocado 0.09 10 Banana 0.06 8 Pear 0.05 12 Tangerine, Florida 0.18 53 Apple, Macintosh 0.0 - Wine, red 0.36 72

Mean 0.09 19

?IUTS Hazel Hickory, meat Hickory, outer shell Hickory, inner shell Peanut Brazil Almond Pecan Walnut

1.92 48 1.96 49 3.38 - 0.0 - 0.0 - 0.0 - 0.0 0.81 20 0.26 -

Mean 0.71

GRAINS AND CEREALS Wheat, seed 0.0 Wheat, Okayama Pref., Japan 0.0 Wheat, grown on forest soil 2.3 Gluten 3.83 Bread, white 0.07 Macaroni 3.33 Oats, seed 1.33 Oats. whole 1.63 Oats; grown on forest soil Oatmeal Rye, seed

0.0 0.12

Rye, grown on forest soil Buckwheat, seed Buckwheat, grown on forest

soil Millet, Hungarian Corn on cob* Corn husks

1.24 0.0 1.98

1.24 0.0 0.57 0.0

Corn meal 0.26 Rice, Japanese, polished 0.82 Rice, American, polished 0.23 Grapenuts 6.03

39

- - 61 -

3 93 33 40 -

3 37 - 59

37 - 16 -

8 23

6 168

Mean 1.10 37

WSCELLANEOUS Tea Cloves, whole Pepper, red, whole* Vinegar, cider

0.0 - 0.28 - 0.06 - 1.36 -


Item

TABLE 4.-continued

(pg/g (!Jg/lOO wet wt) calories)

Item @g/g (!Jg/lOO wet wt) calories)

MISCELLANEOUS (cont’d)

Molasses 0.41 12 INSTITUTIONAL DIET, ONE DAY

Cranberry sauce 0.84 - Rat and mouse diet (rye,

corn oil, dry skim milk) 3.20 82 Cigarette ash 39.50 - Sea water (Northeast Harbor,

Me.) dock 0.0 - Sea water (Biddeford Pool),

Me.) surf 2.4lppb - Resin, used, water softening,

Miami, Fla. 0.0 - Soil, garden* 1.36 - Soil, forest 0.57 -

Breakfast

Content Dinner

Content Supper

Content

Mean Total content

0.02 - 0.015mg 7 0.25 -

0.22 mg 26

1.03 - 0.93 mg 74

0.46 1.165 mg

*Available vanadium soluble in HCl from garden soil where vegetables marked (*) were grown Forest soil from where leaves were collected (Table 6).

An attempt was made to correlate vanadium concentrations with unsaturated fatty acid content (Table 5). No regular relation appeared with iodine number or hexa- decanoic, oleic, linoleic and linolenic content, although vanadium was not associated with ricinoleic acid. The refined or solvent-extracted oils and fats were relatively poor. No constant ratio agreeing with our analyses was found with any of the known triglycerides in corn oil [19]; the ratio of vanadium to trilinolein was about 9.7 m- moles per mole, or about one atom per 32 double bonds, and to all the double bonds in the oil, one atom per 4300.

The absence of such a relationship suggested that naturally-occurring vanadium might be associated with one of the minor constituents of fats and oils, partly removed by processing. Therefore, the presence or absence of sterols and steroids, tocopherol, squalene, carotenoids and phospholipids were examined with respect to the presence of vanadium. Phospholipids seemed to be roughly related [l&20], but the others were not, in so far as known, with the possible exception of the carotenoid pigments.

The evidence that vanadium and phospholipids are associated is tenuous, however:

(1)

(2)

(3) (4)

Vanadium, nitrogen, phosph&us and arsenic are similar chemically. Rats and mice can incorporate arsenocholine into lecithin as arsenolecithin [22], and vanadolecithin is a theoretical possibility. Vanadium oxidizes the double bond in both mammalian liver and soybean phospholipid fatty acids 1231. Refined fats and oils have little phospholipid [19]. Phospholipids in milk mainly remain in buttermilk [24] which might explain the absence of vanadium in butter and its presence in dried skimmed milk. Corn and soybean oils are the largest commercial source of lecithin, which is added as an emulsifier to many foods [19].

Analyses of five commercial lecithins (Table 5) failed to bear out the hypothesis that the largest part of lipid vanadium would be found with phospholipid. In fact, concentrations were relatively low. We must conclude, therefore, that vanadium is concentrated in all but the most refined fats and oils with some unknown substance, but is probably not regularly associated with any specific fatty acid, steroid or vitamin.

1054 HENRY A. SCHROEDER, JOSEPH J. BALA~~A and ISABEL H. TIPTON

Item

TABLE 5. VANADIUM IN PATS AND OILS

Unsaturated fatty acids &g/g (~dloo

wet wt) calories) Oleic Linoleic Linolenic (%) (%) (%)

ANIMAL FATS

Lard, refined Butter, salted Butter, unsalted Cod liver oil refined, Norwegian Mouse Rat Beef bone marrow Pork Deer Chicken Sperm oil Human Human (fed V) Lecithin, bovine 90% Lecithin, egg

VEGETABLE OILS

Castor, refined Safflower, extracted Cottonseed, extracted Sunflower Vegetable shortening Cedar Peanut, pressed Corn, pressed Linseed, crude Olive, pressed Corn oil margarine Soybean, pressed

Mean, edible Rye flour,

Benzene extract Residue Whole

Lecithin, crude soy Lecithin, refined soy Lecithin, vegetable pure

0.0 - 0.0 - 0.0 - 0.0 - 3.06 - 0.06 - 3.17 - 6.76 75 6.82 75

13.85 154 12.67 -

1.62 - 5.83 - 1.28 - 0.0 -

0.0 - 2.85 32 3.02 33 6.95 77 7.49 83 8.98 -

10.87 109 11.05-15.80 112

11.76 - 15.23 169 21.75 273 43.53 484 14.77 164

5.50 0.0 1.24 2.45 1.19 0.58

42 31 - -

44 - 48

38 26 46 - - -

2 0 1.2 1.4

- - - - - - 19 0 - -

8 0 - -

21 0 0 0

10 0 - - - - - -

trace 15 23 21 50 34 53 30 25 65 49 23

7; 48 66

7 33 26 55 19 15 30 55

0 <O.l

0 <O.l <O.l 23

0 0.3

47 0

co.1 8.0

35 48 5.5

Fatty acid content of animal fats from DEUEL [18], of vegetable fats from ECKEY [19 1, and of butter and margarine from BERNFELD et ~2. [21].

Vanadium in vegetation

Table 6 shows the results of a survey of vanadium in leaves of forest trees, shrubs, grasses, fruits and Dutch tulip bulbs. No order was evident. The leaves of most deciduous and evergreen trees showed no detectable vanadium, with the exception of maple, cherry, oak and juniper. The trees were situated in a remote hilltop forest over a mile from the nearest road, and a sample of soil contained 0.57 p.p.m. soluble vanadium. Alfalfa and flax residue were especially rich in this metal. Fruits, seed and berries may provide a source for birds and animals.


TABLE 6. VANADIUM IN VEGETATION

Item (pg/g wet wt) Generic name

LEAVES Poplar Ash, sapling Beech Elm, sapling Basswood, sapling Apple, wild

fruit Maple, sugar Cherry, wild

fruit, with pits Oak, white Pine, white Hemlock, northern Spruce Juniper, spreading, wild Fern, suburban Fern, sensitive, wild

Wood chips, soft wood Barberries Grapes, wild Sumac berries Flax, residue (animal bedding) Clover, field Alfalfa Tulip bulbs: orange

blue black yellow white red

0.0 0.0 0.0 0.0 0.0 0.0 0.78 0.93 0.76 1.27 0.31 0.0 0.0 0.0 0.27 1.16 0.25 0.0 1.25 0.97 0.0 8.08 0.70 4.00 0.0 0.0 0.56 0.21 0.60 0.85

Pop&s tremuloides Pyrus americana Fagus grandifolia Ulmus americana Tilia americana Pyrus ma/us

Acer saccharum Prunus serotina

Quercus alba Pinus strobus Tsuga cana’adensis Picea rubra Juniperus communis

Berberis vulgaris

Rhus typhina

Trifolium repens Medicago sativa

Eflect of vanadium on serum cholesterol of man

CURRAN et al. reported that diammonium oxytartarovanadate given orally to five normal young male volunteers lowered plasma cholesterol significantly (P < 0.05) at the end of a 6-week period [25]. The initial level was approximately 170 mg per 100 ml, falling to about 155 mg. In order to ascertain whether or not this compound acted similarly in older individuals confined to a mental institution, two prolonged experimental trials lasting 6 and 16 months were run on 15 patients, serum cholesterol being measured at monthly intervals. The results are shown in Table 7. The dose, 4.5 and 9 mg vanadium per day, was less than that used by CURRAN, in order to avoid the appearance of ‘green tongue,’ a disturbing symptom especially for the mentally un- balanced. Vanadium appeared constantly in the urine and erythrocytes (Table 8).

Six individuals responded by reductions in circulating cholesterol,usuallytemporary. A 83-yr old man suffered a cerebrovascular accident and his level dropped 47 per cent or more until his death 5 months later; we did not ascribe this effect to vanadium. Values of a 54-yr old man, a 68-yr and 76-yr old woman fell 20-26 per cent below their lowest control levels on two or more occasions, but these drops were not sustained. A 76-yr old man with a control level of 274 mg/lOO ml serum had single levels 7 and 11 months later which were 28 and 29 per cent lower. A 47-yr old man with a control of 243 mg/lOO ml responded well, 7 of 14 subsequent values being 20-36 per cent of the initial and all being 10 per cent or more lower. Other patients did not

1056 HENRY A. SCHROEDER, JOSEPH J. BALASSA and ISABEL H. TI~TON

TABLE?'. CHANGES IN SERUM CHOLESTEROL ASSOCIATED WITH ORAL VANADIUM, CHROMIUM, MANGANESE AND EDATHAMIL CALCIUM IN MAN

No. of patients Age Orr) Initial* Months Change? Dose1 Agent and date (mtO0 given (%) (mp/

d ? Mean Range day)

Nov. ‘58-Jul. ‘59 Vanadium 4 6 72 :z;; 225 6 -3.5 9.0 Control 2 14 72 254 +6.6 Edathamil 0 6 80 (72-85) 247 8 +8.9 1000

Feb. ‘59-Jul. ‘59 Edathamil Control

10 8 71 (43-87) 246 5 -2.0 1000 1 13 77 (61-88) 249 -0.8

Aug. ‘61-Feb. ‘62 Chromium Control Chromium and

vanadium

1 4 76 W-89) 298 6 -9.0 2.0 5 5 71 (53-87) 230 -2.4

4 1 65 (46-77) 245 4 -2.4 2.0+4.5

Feb. ‘62-Jan. ‘63 Vanadium (cont.) 4 1 240 11 -11.15 4.5 Control 2 3 70 (59-79) 224 0.0

Jun. ‘62-Dec. ‘62 Manganese 3 4 75 297 6 -3.4 10 Control 3 3 70 224 +1.0

*Initial level represents the mean of three control values taken two weeks apart. TChange of means of last three. values from control levels. No sex differences were apparent. $Dose as metal except for edathamil calcium. Compounds given were diammonium oxytartaro-

vanadate, chromium acetate and manganese citrate. $Change mainly from two patients. Total change after 15 months on vanadium, 13.5 per cent.

behave similarly. Mean values were relatively unchanged and high ones were less affected than were lower. Statistical analyses by regression coefficients showed no significant differences from control values. No patient lost weight.

The effects of oral chromium acetate, manganese citrate and edathamil sodium were also evaluated, as these substances have been reported to be lipotropic in one way or another [5,26]. A combination of vanadium and chromium was associated with more than a 20 per cent reduction in circulating cholesterol twice in the fifth patient and once in the sixth; in others effects were negligible. Chromium alone was followed by similar depressions twice in one hypercholesteremic woman. Changes during administration of manganese and edathamil were small and wide downward fluctuations did not occur in the control groups. Regression coefficients did not differ significantly from controls. No toxic symptoms appeared.

Turnover of vanadium in man

Vanadium was not found in 15 samples of normal red blood cells but was constantly present in serum (Table 8). When the oxytartarovanadate was given orally it was detected in washed red blood cells in concentrations of 0.37-O. 8 1 p.p.m., while serum values were unchanged from the normal ranges. Corrected to the ‘Standard Man’ and a hematocrit of 45 per cent, 1380 pg of vanadium were circulating in normal serum at the time the blood samples were obtained, or approximately the daily intake. The patients taking 4.5 mg per day had circulating 2300-3350 ug, according to these


TABLE 8. VANADIUM IN HUMAN BLOOD AND URINE

BLOOD* Serum: Mean

Range Erythrocytes; washed

Mean Range Washed Unwashed

Whole blood: Mean

Controls Patients taking V(4.5 mg/day)

No. of subjects @g/100 ml) No. of subjects @g/100 ml)

13 42 4 47 3548

19 0 5 48 37-81

4 37 4 41

23 47.5

URINE Single samples :

Mean Range Excreted

24-hr samples after single dose of 4.5 mg

Excreted Same

Excreted

URINE (Method of ROCKHOLD and TALVI~E [27]) Controls

Mean per month (13 months, 65 samples)

Range Excreted 3 days after 3 weeks after

6 0 16.2 5.3-29.6 s.o%t

3.3 26.0 0.4% 8.0%

25.0 2.7 5.5% 0.9%

15 0 15 0

5 16.9 7.1-28.3 5.37;

17.8 2.9

*Serum analyses were made on 4 single samples and on 2 pools of 5 and 4 samples obtained from laboratory personnel and patients. Erythrocytes were washed with saline and analyses were made on 4 pools of 4-5 samples each. In the cases of the 5 patients taking vanadium, pools of 2-5 samples were analyzed.

tVolume, corrected to 1.4 1 per day owing to difficulty in obtaining 24-hr collections from psychotic patients.

analyses, or about one-third to one-half of the total estimated daily intake. Serum and fat are therefore the main storage depots for vanadium in man. Furthermore, the lack of increase in serum values when vanadium was fed suggests that some carrier was already saturated. A ‘transvanadin,’ however, has not been isolated.

In an attempt to demonstrate a carrier, 15 ml of normal human serum containing 45 ug of vanadium per 100 ml was precipitated with trichloracetic acid. The washed precipitate contained 0.2 ug or 1.33 pg per 100 ml original serum. The supernatant was extracted with acetone and 3 mg of precipitate in TCA isolated; it contained 6.6 l.tg or 43.22 pg per 100 ml of the original serum; that is, 2200 p.p.m. vanadium. None was detected in the acetone extract. Furthermore, all of the serum vanadium was found in the lipid fraction. We conclude that a carrier of vanadium with these solubilities is present in serum, and was some vanadium complex dissolved by the acid from a lipid or lipoprotein.

1058 J~ENRY A. SCHROEDER, JOSEPH J. BALASSA and ISABEL H. T~FTON

No vanadium was detected in 6 normal urines by our method nor in 30 by the method of ROCKHOLD and TALVITE [27] which is less exact. When patients had been taking vanadium, urinary elimination was approximately 5.0 per cent of the amount ingested, with wide individual variations. When a subject received one dose, excretion also varied considerably. When necessary, urine volumes were corrected to 1.4 1 per day as 24hour collections on psychotic patients were usually impossible to obtain.

DISCUSSION We will examine vanadium according to our 6 criteria which help suggest whether

an element has a biological function or is a contaminant [ 15-17,28,29].

(1) Ubiquity

(a) On the earth. Vanadium is an ubiquitous element on the earth’s crust, ranking 10th in abundance of the 29 trace metals in which there may be biological interest. There is more of it than of the essential metals, zinc, copper, cobalt and molybdenum and the tissue-accumulating metals, cadmium, lead and tin; the average concentration in the geosphere is 110 p.p.m. [30]. It ranks 10th in abundance in the universe, 13th in sea water, and, according to our results, 10th (0.3 p.p.m.) in the body of man with a predilection for fat, serum, bone (where concentrations have reached 1.5 ug/g, wet weight) and teeth*.

Natural vanadium occurs in igneous rocks, in titaniferous magnetites, in certain deposits of phosphate rock, in shales, in some uranium ores and in asphaltic deposits on all continents. Metal vanadates are found in many base metal deposits. There is little vanadium in most marine phosphorites [I], although those of Montana, Wyoming and Idaho have much. Soils vary considerably in content, but it is usually present in appreciable amounts [4, 301.

(b) In water. MITCHELL did not find any vanadium in waters of the United States, Argentina or Germany [31]. It has been detected, however, at times, in 22 per cent of 59 analyses of 15 major rivers of the United States and Canada, appearing in 9, more or less seasonally, at levels of 6 p.p.b., especially in the Hudson and St. Lawrence Rivers [32]. DURFOR and HAFFTY found it in 36 per cent of municipal water supplies of 42 United States cities, in concentrations of l-6 p.p.b. or more; occasionally values were higher 833. The National Water Quality Network [34], however, detected it in only 2 of 119 analyses at 71 locations on 28 major rivers, both in the Southwest, by probably less sensitive methods, and their latest survey showed it in only 11 of 142 municipal waters [35]. The Colorado Plateau contains vanadium in uranium ores, which is expressed by its presence in waters supplying cities in California and New Mexico (where one sample contained 70 p.p.b.). BERTRAND concludes, from most of the older data, that there is very little vanadium in most natural waters, although some mineral springs have it [4]. More recent surveys agree.?

It is obvious that drinking water, with rare exceptions, could supply only a few micrograms of vanadium per day, as compared to the milligram quantities in most vegetable foods. Where vanadium was found in water, the calculated daily intake was approximately 0.2 per cent of that in food, with one extreme value of 3.5 per cent. *Vanadium was found in high concentrations in the callus and new bone growing about experimental fractures in dogs [86].

STANK and STORVICK, however, found vanadium by the copper spark spectrographic method in 145 potable waters in Wyoming, at 0.034.22 p.p.m. [87].


Estimates of vanadium in sea water vary from undetectable amounts to the 0.3 p.p.b. found by GOLDSCHMIDT [30] and the 2.4-7 p.p.b. listed by MACON [36] in Harvey’s table. We found 2.47 p.p.b. in surf water but none in harbor water. Marine muds, however, are often rich in vanadium, suggesting that the metal in river water is deposited in the sea in a more or less insoluble form as probably is titanium [17]. River mud also contains vanadium in concentrations similar to those in the lithosphere

[371. (c) In air. TABOR and WARREN [38] found vanadium in the air of most United

States cities. Of 754 samples, 46.6 per cent had 0.002-O. 02 ug/m3; 36.9 per cent had 0.02-O. 2 ug/m3 and 4 per cent 0.2-O. 6 ug/m3. Jersey City, N.J., had the highest level and Fort Worth, Texas, the lowest. Boston, New York, Philadelphia, Portland, Oregon and Washington had high values, while some industrial areas such as Chattanooga, Denver, East St. Louis, Houston, Louisville, New Orleans and Tampa were fairly low. Suburban sites had unusually high proportions of samples with quantities above the minimum detectable amount, while the largest number with less than detectable levels (0.002 ug/m3) were of urban origin. When present, about 132 ug are in the lungs of ‘Standard Man,’ who breathes 20 m3 of air per day and retains 123 per cent of particulate matter inhaled [39]. A vanadium concentration of 0.01 ug/l. would provide 13 ug per year of which 9 ug would be deposited in the lung, more than enough to account for the pulmonary content. The absence of detectable vanadium in the lungs of 42 per cent of Americans is consistent with a non-uniform distribution in the air.

Petroleum usually contains large amounts. The hypothesis that pulmonary vanadium may be partly dependent upon the use of fuel oil for heating is suggested by the following coincidences: (1) The lowest levels and lowest prevalences of pulmonary vanadium were found in samples from two Southern cities, the highest from Northern ones (Table 1). (2) The lowest values in air came from Southern cities, the highest from Northern ones [39]. (3) Prevalence in foreign lungs was greatest in Switzerland and the Middle East where oil is widely used. The lungs appear to be a minor portal of entry of vanadium into the body. How much is further abeorbed is unknown.

(2) Presence in living things

(a) Plants. Vanadium has been universally found in coal ash and in petroleum, bitumins and asphalts. Some samples of petroleum ash are extremely rich (50-75 per cent of VaO5) and the metal has been recovered commercially from it [30]. Vanadium porphyrins have been detected, indicating biological origin of bitumins and petroleum.* Spectrographic analyses of sands and muds are useful in prospecting for vanadium- bearing oils.

All living plants examined, according to BERTRAND [4], have shown concentrations of 0.01-0.87 p.p.m. wet weight, 1 p.p.m. dry weight and 7.1 p.p.m. ash weight.?

*Vanadium and to less extent, molybdenum and nickel are associated with petroleum and bitumins and are effective catalysts for the synthesis of hydrocarbons from biogenic fatty acids deposited on the anaerobic ocean floor in dead bodies of marine organisms, probably plankton. These elements may be present as organo-metallic compounds and, with bacteria, may catalyze the fission, con- densation, cyclation and dehydration of fatty acids into petroleum [36].

tin this discussion, analyses for vanadium by emission spectroscopy using carbon electrodes containing vanadium are omitted when known. Wood contains vanadium [4], although we did not find it in soft wood chips.


DEAN GUELBENZU and his colleagues [40] detected vanadium only infrequently in a number of Spanish foods: legumes, none; cereals, 50 per cent; condiments and spices, 12 per cent; grains, 40 per cent; fruits, rarely; vegetables, 15 per cent. Concentration in the soil is not known.

The fly mushroom Amanita muscaria is an accumulator from soil, concentrating vanadium to 61-181 p.p.m. dry weight; no other Amanita, fungus or plant examined had this property [4]. Root nodules of beans and lupins (which contain nitrogen- fixing bacteria) concentrate it several times more than the aerial portions. Our results on plants are largely in accord with these older data. We may, therefore, conclude with BERTRAND [4] that vanadium is almost constantly present in plants, but may be absent from the leaves of trees.

(b) Animals. Most marine animals analyzed have contained small amounts of vanadium [41], mean about 0.28 p.p.m. wet weight. Fatty fish like sardines and herring had 0.2 p.p.m. but the flesh of larger fish failed to show any [42]. The pleobranch ascidians are efficient accumulators of vanadium; their blood has up to 4 per cent vanadium [43] and contains free sulfuric acid with a pH of 1.5 [43, 441. Niobium, chromium, titanium, manganese and iron, as well as vanadium, are also concentrated by some tunicates [44]. The vanadium, which probably is extracted from mud, occurs in the blood in vanadocytes.* Obviously the vanadium-sulfuric acid mechanism is not too efficient, although sufficing for tunicates. Ascidians represent an evolutionary regression, an experiment which came to a blind end probably because of the chemical limitations of vanadium as a major catalytic constituent of living things. Their larvae have a notochord and they are related to our common ancestor, Amphioxus, degenerat- ing therefrom to the modern sea squirt who has only a single ganglion for a nervous system.

The picture in vertebrates and especially mammals was less clear. BERTRAND [4] stated that the liver contains the most, finding it in that of dogs and deer, but not in mice. One of us (I.H.T.) failed to detect it in 4 Tennessee deer livers and kidneys by emission spectroscopy. DANIEL and HEWSTON [47] were unable to show it in foods, eggs, or rats and believed that vanadium, if present in mammals, occurred in amounts too small to be detected by sensitive methods (1 p.p.m. ash or less). Our data do not bear out this idea, but show that there are wide variations in absorption and retention from species to species and organ to organ, with a common concentration in fat.

The finding of vanadium in fats and oils has partly clarified some of our conflicting data and those of others. Seeds, which contain lipid [19] had moderate quantities and a flax residue had much. Relatively large amounts occurred in alfalfa and legumes which are dependent upon nitrogen-ting bacteria. Its presence in gelatin, deer hoof and human hair suggest an affinity for keratin. Although common denominators are not wholly evident, some order for the presence or absence of vanadium is vaguely discernible.

*Vanadocytes are green blood cells containing hemovanadin, a pigment composed of pyrrole rings, probably in a straight chain comparable to those of bile pigments, and a protein, possibly a primitive analogue of hemoglobin. Virtually all of the vanadium in blood is found in the vanadocytes. Their function is not known, although hemovanadin is a strong reducing agent. WEBB [45] did not believe that they carried oxygen. HENZE [43], who first found vanadium in tunicates in 1911, thought that it might be a reducing agent for carbonic acid. Vanadium in vanadocytes exists in the trivalent form as Vn(S04)3 1461.


In man the situation is more clear. Vanadium was found in fat and serum but not regularly in any other tissue. Variations in organ content could depend, therefore, on the amount of fatty infiltration in the tissue analyzed. In the case of wild animals, no order dependent upon fat content appears, except for bone marrow. According to spectrographic analyses of fat-free organs, vanadium ranks 25th of the 29 metals of interest, with the ‘Standard Man.’ having less than 0.1 mg in his whole body [39]. This estimate is erroneous.

(3) Atomic structure and essential actions

Vanadium occurs in 5 valence states, four under natural conditions. Other than as an alloy metal, its widest application to industry lies in the catalytic properties of its compounds. It is used for oxidation, dehydrogenation, dehydrocyclization, hydroxy- lation, hydrogenation and hydrogenolysis [l]. Some compounds have unpaired third electrons and have magnetic properties. Vanadium pentoxide has been used as a catalyst for preparing sulfuric acid since 1900. Some 18 other catalytic uses are known and many metallo-organic compounds have been prepared [l]. Vanadyl linoleate is an excellent drying agent for paints.

Vanadium forms somewhat labile chelates, bonding to oxygen in higher valence states and to nitrogen in lower. It is a strong reducing agent. Its magnetic moments are close to those of molybdenum, chromium and manganese in coordination numbers 4 and 6. Some of its properties are similar to those of arsenic and phosphorus.

TANNER [48] examined the ability of transitional metals to oxidize several biological substances in model systems. Sodium metavanadate was considerably more active than the other metals on glyceraldehyde and epinephrine and was uniquely effective on norepinephrine, while cobalt oxidized linoleic acid and manganese oxidized hydroquinone. Metal specificity was not obviously demonstrated on ascorbic acid or cysteine. TANNER further examined the mechanisms of metal-catalyzed oxidations and their inhibition by drugs. Ascorbic acid considerably enhanced the vanadium- norepinephrine reaction, while several complexing agents partly inhibited it. Vanadium oxidized linoleic acid to some extent.

Shortly after TANNER’S work appeared, MARTIN et al. [49] reported similar model oxidations of 5_hydroxytryptamine, 5-hydroxytryptophane, 5-hydroxyindole acetic acid, 3,4_dihydroxyphenyalanine (DOPA) and 3,4-dihydroxyphenylethylamine (Dopamine) as well as confirming the action of vanadium onepinephrineandnorepine- phrine. Therefore, in so far as is known, mono- and ortho-dihydroxy indole and phenol compounds appear to be sensitive to oxidation by vanadium, a reaction in line with its affinity for hydroxyl groups and its binding to oxygen in the test tube and on the earth’s crust. Model experiments indicating preference of a specific metal for a biological compound suggest the possibility that the metal may be a natural cofactor in a biological reaction involving the compound.

(4) Homeostasis

All of the essential trace (and bulk) metals are controlled in mammals by homeostatic mechanisms, either demonstrated (iron, copper, manganese) or implied (zinc, molybdenum, cobalt, chromium). These mechanisms prevent excessive accumulations or depletions in the face of variations in oral intake and provide optimum tissue concentrations during life. The newborn human being is usually supplied with

1062 Emmy A. SCHROEDER, J-H J. BALASSA and ISABEL H. TrpTo~

adequate or even excessive amounts, there being no placental barrier to transfer from maternal blood, and milk provides a continuing supply. We can make a reasonably valid assumption that a metal not found in the newborn or in milk is probably not essential for life or growth. Newborn fat has not been examined for vanadium.

Pulmonary accumulations in an environment where air-borne metal is present are no indication that a metal fulfills a function. Aluminum, lead, barium, tin, vanadium, titanium, nickel, chromium, cadmium and bismuth have been found in the air of United States cities and suburbs and likewise in the lungs of Americans. Therefore, pulmonary vanadium can be discounted as having an essential metabolic role.

The oral intake of vanadium is larger than that of any ‘abnormal’ metal we have examined, except tin, although less than iron, manganese or zinc. It approximates that of copper. The high oral intake, low urinary excretion, high serum levels and accumulation in fat, but in no other tissue, suggests that powerful regulatory mechanisms exist. When fed as a soluble complex to patients the lack of change of serum levels, the small proportion excreted in urine* and the storage in fat and in erythrocytes indicate that the human body has a homeostatic mechanism for vanadium which can be partly overcome by large doses. The situation is reminiscent of that for copper, iron and manganese. The obvious routes of excretion to be explored, as with other trace metals, include bile, intestine and pancreatic juice.

Vanadium is not a toxic trace metal for man. We have fed patients 4.5 mg per day as the oxytartarovanadate for 16 months with no signs of intolerance appearing. SOMERVILLE and DAVIES [51] gave each of 12 patients 13.5 mg per day for two weeks and then 22.5 mg for 5 months; gastrointestinal symptoms appeared in 5 and green tongue in 5. DIMOND et al. had essentially the same experience with 4.5-18 mg per day for 6-10 weeks [50]. Vanadium was found in the urine in increased amounts in all three series. The lethal oral dose for man is not known.

Inhaled vanadium dusts cause pulmonary irritation but little more. LEWIS effectively disproved that a long series of varied disorders in vanadium workers reported in the older literature were produced by vanadium [2]. Cough, sputum, wheezing, irritation of mucous membrane, ronchi, injected pharynx and green tongue were found, but there was no evidence of chronic intoxication or injury. Exposed workers excreted 4 times as much vanadium in urine as did their controls [2]. VaOs in aqueous solution is an acid, pH 3, and when inhaled, undoubtedly contributes to upper respiratory irritation, regardless of inherent toxicity, as STOKINGER pointed out so clearly [52].

Large oral doses in laboratory mammals have caused death from gastrointestinal irritation, as might be expected. For the rat, however, the amount required in the diet was 160 p.p.m. Intravenous and subcutaneous toxicity for rabbits and guinea pigs depend upon the compound used, the minimal lethal dose varying from 1 mg to 20 mg/kg body weight; likewise, subcutaneous toxicity for rats varies from 6 to 100 mg/kg. Vanadyl sulphate is the least toxic compound and ammonium metavanadate the most toxic [52]. Rats taking 11 p.p.m. of selenium died more quickly when also

*Very large doses of diammonium oxytartarovanadate (9-22.5 mg vanadium per day), however, have been reported sometimes to cause high urinary levels. D~orm et al. found a very variable excretion, from 10-70 per cent of the amount ingested, after patients had been taking it for some time [50]. These urinary levels are higher than ours (Table 8).


taking 5 p.p.m. vanadium in drinking water [53]. TALVITIE and WAGNER [54] studied the effect of injected pentavalent vanadium salts

in rats and rabbits and believed that 0.25 mg/kg given repeatedly may have been slightly nephrotoxic to some animals. Renal excretion was rapid, 61 per cent being eliminated in 24 hr, while 10-12 per cent was excreted by the intestinal tract and most of the remainder was deposited in the skeleton. Injected vanadium salts caused vasoconstriction in rabbits [55] and rats [56].

To less complex living things, soluble vanadium was only slightly toxic; 10-20 p.p.m. to soybeans; 26 p.p.m. to beets; 40 p.p.m. to barley; 20 p.p.m. to wheat and 22 p.p.m, to oats. The effects varied with the compound. Ascidians, of course, tolerate several hundred parts per million.

(6) Biological eficts

No other trace metal has so long had SO many supposed biological activities without having been proven to be essential. The definitive experiment has not been done. BOKORNY [57] in 1904 showed its lack of toxicity for many bacteria at levels of 1300- 2600 p.p.m. As quoted by BERTRAND [4], FROUIN and his co-workers stated that it increased the growth of Pseudomonas pyocyaneus, tubercle bacilli and Aspergitlus niger at low concentrations. In the 1950’s a number of workers, especially BORTELS (quoted in [4]), demonstrated that azobacteria could use vanadium for the fixation of nitrogen (although molybdenum is the usually accepted necessary element); the optimum concentration was 0.01-0.25 p.p.m. In 1942 HORNER et al. [58] proved conclusively that vanadium from trace levels to 1 p.p.m. increased the fixation of atmospheric nitrogen by 9 strains of Azobacter 5-25 times, that there was almost no nitrogen fixation and a reduction in the consumption of glucose in its absence, and that molybdenum had a similar action.

D. BERTRAND [4] showed that Aspergillus niger responded to vanadium at 0.04 p.p.m. by increased growth. No proven response of higher plants has been demonstrated, except those dependent upon Azobacter, although in the cases of red clover and barley, vanadium follows Gabriel Bertrand’s Law of optimum nutritive concentration.* Finally ARNON and WFSSEL [59] showed that it was essential for optimum growth of the green algae, Scenedesmus obliquus, being concerned with photosynthesis under strong light [60].

In animals effects are less understood. D. BERTRAND [4] stated in 1948: “We are completely ignorant of the physiological role of vanadium in animals, where its presence is constant.” Many attempts have been made to prove one. BERNHEIM and BERNHEIM’S classical papers [23, 611 demonstrated oxidation of brain, liver and soy phospholipids by mammalian liver suspensions in the presence of IO-20 pg of vanadium. Two effects were shown, dehydrogenation of fatty acids to cause unsaturation and oxidation at double bonds to fragment the chain. The vanadium-protein system was inhibited by manganese and partly by cobalt. Lately interest in its possible role in lipid metabolism has revived. MOUNTAIN et al. [62] showed that dietary vanadium lowered hepatic, plasma and other tissue phospholipids in rabbits. SNYDER and CORNATZER

*Gabriel Bertrand’s Law, formulated in 1903, states that absence of a metal essential for plants causes death of the plant, while an excess is injurious. He proved this Law for manganese in 1905 [4]. The Law requires some amendment in regard to the calcium concentration in the environment, which can markedly influence absorption of metal ions by both plants and animals.


[63] indicated that hepatic synthesis of phospholipids was decreased and oxidation augmented by vanadium. CURRAN et al. [5] clearly demonstrated a marked decrease in the hepatic synthesis of cholesterol and fatty acids in rats (and increased synthesis with chromium and manganese), a decrease in the aortic content of cholesterol-fed rabbits [6], a slight lowering of plasma cholesterol in young men [25] and a decreased synthesis of cholesterol in human brain tumors [64]. According to our concepts, however, inhibition of a system by a metal ion is no sine qua non of essentiality (cf. cadmium [ 15]), although oxidation may be.

PERRY et al. [7] found that the activity of guinea pig liver monoamine oxidase with tryptamine as a substrate was considerably enhanced by 1 .O mM V3+ and V4+, but not by V’s+ nor by any other transitional metal of the first group. Detailed data have been published elsewhere [65]. One of us (H.A.S.) has suggested that tissue monoamine oxidase might contain vanadium as a co-factor [65], for model experiments previously cited are curiously consistent in this respect.*

LEWIS tested this idea in the dog by injecting NaV03. Urinary excretion of 5- hydroxyindole acetic acid, a metabolite of serotonin, was decreased by large doses, indicating that no enhancement of enzyme activity occurred in viva, but with smaller doses excretion was moderately increased. LEWIS states that the results were not too clear-cut [67].

The site of inhibition of biosynthesis of lipids may be at two places. AIYAR and SREENIVASAN [68] showed that injected vanadium decreased rat hepatic coenzyme A, coenzyme Q and succinoxidase, an effect counteracted by simultaneous injection of the component parts of the CoA molecule. They believed that it acted on the synthesis of CoA, possibly through an effect on sulfhydryl groups and adenosine triphosphate reserves, for both of which it has strong basic affinities. AZARNOFF and CURRAN [69] on the other hand, demonstrated inhibition of cholesterol biosynthesis between the stages of mevalonic acid and b-methyl crotonate. Mevalonic kinase requires manganese as a cofactor [70]. WRIGHT et al. [71] suggested that oxidative phosphory- lation was inhibited. WHITEHOUSE et al. [72] found no effects in vitro on the oxidation of cholesterol, nor were bile acids in human feces increased. CURRAN’S experiments suggest that vanadium may mobilize tissue sterols while it depresses biosynthesis of cholesterol in man.

Vanadium at 5 p.p.m. inhibited growth of tubercle bacilli markedly [73]; this action was opposed by chromium and manganese. Furthermore, dietary vanadium decreased both the mortality of tuberculous mice and the pulmonary lesions of rabbits, apparently affecting some host factor [74]. The total evidence, therefore, is impressive that vanadium has a biological role, especially regarding lipid metabolism, and that vanadium-manganese or vanadium+hromium antagonisms exist in several systems.

Another indication of a biological function concerns its effect on caries and the formation of dental enamel. RYGH [75] found marked stimulation of mineralization of bones and teeth by vanadium salts, while caries was prevalent in vanadium-deficient rats and guinea pigs. GEYER [76] confirmed RYGH’S findings in hamsters, using both oral and subcutaneous vanadium, which inhibited the development of caries in spite of a cariogenic diet. Vanadium is isomorphous with phosphorus and might replace it in

*Plasma monoamine oxidase, however, has copper as a cofactor [66].


apatite [77]. Human caries need re-examination from this viewpoint.* Observations pointing superficially to the lack of essentiality of vanadium are no

longer valid (1). It was not detected in most human tissues by spectrographic methods, which can pick up 1 p.p.m. ash or 0 .Ol pg/g wet weight. These tissues, however, were dissected free of fat [12]. (2) It was not found in many tissues of normal rats [47]. (3) Little was supposedly absorbed by man. (4) Many of its effects in vitro and in viva involve inhibition (Table 9).

TABLE 9. SUMMARY OF BIOLOGIC ACTIONS OF VANADIUM

System Effect Concentration Antagonist Reference

HEPATIC SYNTHESIS OF : Cholesterol Fatty acids Phospholipids Coenzyme A

HEPATIC OXIDATION OF : Phospholipids

GROWTH OF : Tubercle bacilli Azobacteria (and dependent

plants) Aspergillus niger Scenedesmus obliquus

Chilomonas paramecium Monoamine oxidase activity Mineralization of teeth Development of caries

ACCUMULATED BY: Amanita muscaria Phlebobranch ascidians Petroleum Vegetable oils Animal fats

Depressed Depressed Depressed Depressed

Increased

Inhibited

Essential (or MO) Essential Essential

(Photosynthesis) Increased Increased Increased Inhibited

0.1 rnM 0.1 rnM in vivo in vivo

l/SO

5 p.p.m.

0.1 p.p.m.

0.1 p.p.m.

c51 p.p.m. 1.0 rnM in vivo in vivo

150 p.p.m.

Cr, Mn Cr, Mn

[51 [51 KG 631 [681

Mn, 0)

Cr, Mn

~3, 611

[731

t581 [41 [59, 601

[41 t71 1751 [76, 871

141 1000 p.p.m. dry Up to 70% ash 345 p.p.m. 7-14 p.p.m.

[41 [301

Balance qf vanadium in man The intake and turnover of vanadium in man can be approximated from these data.

PERRY and PERRY found less than 8 pg per day in the urines of a group of 24 normal persons in St. Louis by a spectrographic method [78]. The maximum concentration was 22 pg/l.; IO samples were too dilute for accurate assay. Analyses of the urines of a single young man indicated that the total excretion was independent of the volume and was little affected by pH. The PERRYS’ two sons excreted vanadium at less than a fifth of the concentration of their grandfathers’ urines. The metal was detected readily in only a third of 12 African patient’s urines. Our chemical method detected none in urine, possibly because of methodology, possibly because of regional differences in intake.

We can construct an approximation of daily human balances, based on dietary surveys and using a ‘well-balanced’ diet:

*Some inhibition of caries was found by TANK and STORV~CK in areas where vanadium in water was high, independent of fluoride or selenium content. Fluoride, however, appeared to exert a greater effect than vanadium [87].

1066 HENRY A. SCHROEDER, JOSEPH J.

Intake (pg) In food: 2000 (lOOO4000) In water: O-8 In air: CO.2

BALASSA and ISABEL H. TIPTON

output bd ln urine: In feces: EO Retained in lung: O-O. 2

Calculations from Tables 4’and 5 show clearly that several types of dietary habits will influence the intake of vanadium: (1) A vegetarian diet will usually result in higher intakes than a non-vegetarian one; (2) A diet high in unsaturated fatty acids from vegetable sources will result in higher intakes than one using saturated fats processed froin animal sources; (3) A large caloric intake from wheat flour can cause a low intake of vanadium, particularly when most of the protein comes from animal sources (meat, gravy, bread, butter, fruit and lard would have almost none); (4) Certain special diets, such as the ‘Prudent Reducing Diet’ of JOLLIFFE et al. [79] are low in vanadium (200-300 pg) unless corn oil is added, 45 ml of which would supply an extra 500 pg; (5) The major determinant of intake is the type of fat in the diet.

Based on our analyses of fat and serum, estimates of the total bodily content of vanadium must be substantially revised. Prior estimates of < 0.1 mg excluded these two tissues [14,39]. The ‘Standard Man’ has 10 kg fat and 3 kg serum [39]; therefore, the body pool can be conservatively calculated as follows:

Tissue &gig) Total (mg) Fat 1.62 16.2 Serum 0.35-0.45 1.38 Bones, teeth and bone marrow <1.5 < 15.0 [39] Other organs <O.l [39] <O.l [39]

Total 0.3 17.5&42.68

Without bone, this amount places vanadium among the prevalent trace metals, approximately equal to manganese (20 mg) and much ahead of chromium, molybdenum and cobalt.

Our normal serum levels suggest that the transport of vanadium is by a carrier not present in precipitated protein. TALVITIE and WAGNER [54] found 0.9-l . 1 pg/g in the blood of two rabbits injected daily with sodium metavanadate at 0.25 mg/kg, a concentration similar to that measured by us in human subjects taking vanadium orally (0.84-l .28 pg/g). If ‘transvanadin’ exists, it must be present in appreciable quantities and be relatively easy to isolate.

Furthermore, if the total body fat content of vanadium is as large as we calculate, it is likely that human beings losing weight will excrete considerable amounts in their urines, 1.6 mg/kg of fat lost. If we neglect variations in different fats, the patient given it may have accumulated 60 mg. No other available tissue stores of vanadiumare known.

Eflect on circulating cholesterol in man In addition to the initial report of CURRAN et al. on lowering circulating cholesterol

in young men [25], LEWIS studied 32 vanadium workers over 40 years of age, com- paring them with 45 age-matched men from the same area. Those exposed and excret- ing more than normal amounts of urinary vanadium had slightly lower cholesterol


levels than their controls (by 26 mg and 20 mg/lOO ml, P-CO .05). Several of his workers showed ‘green tongue.’ Mean control values were 230.9 mg and 226.7 mg/lOO ml [80]. On the other hand, SOMERVILLE and DAVIES recently reported no change in 12 hypercholesterolemic patients given diammonium oxytartarovanadate for 6 months; mean control level was 411 mg/lOO ml and the mean age 49.2 yr [5 11.

DIMOND et al. gave the same drug to 6 patients for 6 to 10 weeks; temporary falls in cholesterol occurred in two and there were no significant changes in phospholipids, triglycerides, or urinary steroids [50]. Our experience is essentially the same, except that we gave smaller doses for longer periods and most of our patients were elderly.

These five experiments indicate that vanadium’s effect on cholesterol in man is slight. In view of normal dietary intakes and serum levels, it is probable that the slight effects observed are pharmacological and not caused by correction of a physiological deficiency. We must point out, however, that dietary regimens based on the forced feeding of unsaturated fats, which lower plasma cholesterol in man, are associated with high intakes (l-4 mg/day) of vanadium and that the feeding of vanadium-poor saturated fats raises cholesterol. It would be rather interesting if the present controv- ersy on the intake of polyethenoid fats and atherosclerosis were resolved by the finding that a ratio of vanadium to fat was a factor in circulating cholesterol homeostasis. Soybean lecithin has been reported to lower circulating cholesterol in man [81] and to protect rabbits from experimental atherosclerosis [82], effects which could depend upon unsaturated fatty acids, vanadium or both. At present, however, we must conclude that vanadium, the other two lipotropic metals found by CURRAN [5], and a metal-chelating agent [83] have no sustained effect on circulating cholesterol levels in older patients, those who may need it most. This conclusion could have been made previously if the intake and tissue stores had been known.

Availability of vanadium for the biological cycle

Not much is known about factors which influence uptake of vanadium by plants. During weathering, the prevalent trivalent magmatic insoluble vanadium is oxidized to the quinquevalent state, which forms soluble vanadic acid and salts of heavy metals. In 10 New Jersey soils the concentration varied from 11 p.p.m. to 119 p.p.m.; corn leaves took up 0.37-l . 1 p.p.m. vanadium without reference to soil concentration [84]. HANNA and GRANT [85] found 0.8-3.7 p.p.m. dry weight in 9 ornamental plants grown on the same soil; differences in foliage from two sites with identical calcium, but differing hydrogen ion concentrations were not significant. There are undoubtedly some regional differences in available metal, although vanadium-deficient soils have not been demonstrated to our knowledge. Our two soils varied in soluble metal. Although little is known of the availability of plant (or water) vanadium for mammals, it is difficult to believe that the large amounts in plants are all in insoluble forms, and are not absorbed and excreted by a transport mechanism providing constant serum levels.

Essentiality of vanadium

Is vanadium an essential trace metal? Although that question has not been definitely answered, it behaves like one for the following reasons :

(1) It is ubiquitous on the geosphere. (2) It is present in all plants and all animals are exposed to it; it is found in all


crude fats and oils in high concentrations. It is present in human serum and fat.

(3) It has many biological actions in vitro and in vim, especially on lipid metabolism and possibly on the formation of dental enamel.

(4) It has a low molecular weight, is an excellent catalyst, has the proper atomic structure, is a transitional metal, forms chelates, and is chemically suitable for biological functions (apparently performing at least three in the biosphere).

(5) It has a low order of toxicity to most living organisms, and especially to mammals orally.

(6) Mammalian homeostatic mechanisms are implied by serum levels, excretion rates and fat stores. Thus, the evidence is strong. Contrariwise, many of its biological activities are inhibitory. If vanadium performs a necessary or desirable function in man, it probably acts on lipid metabolism.

SUMMARY AND CONCLUSIONS A survey was made of vanadium in human tissues from several parts of the world

and of sources and exposures in food, water and air. Analyses of tissues of wild and domestic animals and of forest foliage were also made.

Vanadium has an affinity for all natural biogenic fats and oils. It was found in vegetable oils, animal fats, human fat and human serum, often in high concentrations. Four highly processed fats and oils had none detectable.

Human organs and non-fat tissues contained little or no vanadium. Half the lungs and a sixth of the intestines examined had it in low concentrations, but it was detected only rarely in other tissues. There were geographical variations in pulmonary vanadium in the United States and in pulmonary, renal and hepatic vanadium around the world. The hypothesis that pulmonary vanadium might depend upon the use of fuel oil was suggested. Organs of several wild animals showed higherthanhumancon- centrations, while domestic animals had it only in fat, at several times human levels. It was not detected in mammalian muscle.

Vanadium was irregularly present in foliage, but was found in most vegetable foods, often in relatively large amounts. Cereals and grains contained an average of 1.1 p.p.m. ; vegetabies 1.5 p.p.m.; nuts 1.2 p.p.m. and fruits little or none. All in one grain was in the fatty portion. The oral intake of man was of the order of 2 mg daily, of which none appeared in the urine. More than half this amount was constantly present in circulating serum. Normal levels were 35-48 c(g/lOO ml. A serum fraction containing 2200 p.p.m. was isolated. The total body pool has been calculated as 1743 mg, prior estimations of < 0.1 mg being erroneous. Powerful homeostatic mechanisms in man were implied.

Feeding experiments with an organic complex in human beings demonstrated that 5-6 per cent was excreted in the urine, that normal serum levels were not altered but that red blood cells and fat accumulated it. No consistent effect of oral vanadium on circulating human cholesterol was shown in 16 months of continuous administration, nor of chromium, manganese or edathamil calcium.

Vanadium behaves like an essential trace metal. It is ubiquitous, is present in plants especially in their oils, and in animal and human fat, has the requisite atomic structure for a catalyst and oxidizes a number of biologically active compounds, is quite non- toxic, is essential for several primitive organisms, is constantly present in human serum


and shows many biological activities in viva and in vitro, especially as regards lipid metabolism in plants and animals. Final proof of essentiality for mammals is still lacking.

There is no reason to believe that serious deficiencies of vanadium exist in balanced diets of human beings. Most diets high in unsaturated fats carry with them large amounts of vanadium. Some factor at present undisclosed governs vanadium homeostasis in man.

Acknowledgments-The authors are indebted to Dr. R. C. ELLINGSON for generous supplies of the vanadium compound used in patients, to Mr. W. H. VINTON, Jr., for the samples of foliage and wild animals, and to Mr. RAY I. PESTLE for the pasture herbage. The technical assistance of Mrs. PHYLLIS ILL~NGWORTH and Miss PAT~UCIA SCARLETT is gratefully acknowledged.

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abnormal trace metals in man-vanadium* · abnormal trace metals in man-vanadium* henry a....

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