Distribution of plasma-bound hydroxyproline in breast cancer, benign breast disease and healthy...

The British Journal of Surgery Vol. 66 : No. 7 : July 1979

Br. J. Surg. Vol. 66 (1979) 449-452

Distribution of plasma-bound hydroxyproline in breast cancer, benign breast disease and healthy females M. F. N E H L A W I , A . C U S C H I E R I , J . C L A R K A N D R. A. B. WOOD*

SUMMARY In normal adult females, patients with benign breast disease and patients with breast cancer, hydroxyproline (OHPro) was found in the plasma attached to the protein jractions which were separated by Sephadex gel filtration. In addition, a distinct moiety, probably a protein sub-unit, containing substantial amounts of this imino acid, occurred in between gel filtration fractions 111 and IV. Quantitative differences in the OHPro content of these various fractions were observed in the groups of' patients studied. Signijicant elevations in the peptide-bound OHPro (fraction I V ) were found only in patients with breast cancer and the level appeared to correlate with the degree of advancement of the disease. Similar findings relate to the OHPro content of the sub-protein moiety which is found between gel filtration fractions III and IV. A reduced level of high molecular weight hyproprotein (fraction I ) was found in the breast cancer group.

URINARY hydroxyproline (OHPro) determination has been used as a sensitive index of collagen metabolism in human tissue (Prockop, 1963). Excretion of this imino acid is elevated in a number of disease states particularly in disorders of connective tissue, skin and the endocrine system (Ziff et al., 1956; Benoit et a]., 1963; Brunish and Sarensen, 1965). An increased excretion of urinary OHPro is also encountered in primary and secondary tumours of bone (Platt et al., 1964). In 1973 Cuschieri reported that urinary OHPro was persistently elevated in patients with breast cancer who subsequently developed osseous deposits and this was later confirmed by Powles et al. (1975) and Roberts et al. (1975).

Plasma OHPro is found in the free and bound forms. The presence of dialysable OHPro-containing peptides in normal plasma has been previously reported (Prockop et al., 1962; Smiley and Ziff, 1964). In addition, there is a form of OHPro which is non-dialysable and protein-bound and which is released by acid or alkaline hydrolysis (Le Roy et al., 1964). The latter has been referred to as hyproprotein or collagen-like protein as it is resistant to hydrolysis by pronase and chymotrypsin. Increased levels of hyproprotein(s) have been reported in a variety of clinical states (Le Roy and Sjoerdsma, 1965). This study was undertaken to investigate the distribution of peptide- and protein-bound OHPro in the plasma of patients with benign breast disease and patients with early and advanced breast cancer. 3 1

Patients and methods Included in the study were 32 patients with histologically proved breast cancer, 14 patients with benign breast disease (fibroadenosis, cystic mastopathy, fibroadenoma) and 24 adult healthy females who were the control group.

On the basis of our method of staging, the breast cancer group was subdivided into 15 with early disease (group A) and 17 with advanced cancer (group B). Patients in group A had no evidence of dissemination and either no histological axillary node involvement or a maximum of two lymph nodes involved. The advanced breast cancer group had either evidence of metastatic spread or advanced locoregional disease with extensive axillary node involvement, defined as three or more ipsilateral axillary nodes with tumour deposits on histological examination.

The assessment of patients with breast cancer included clinical examination, mammography, aspiration needle cytology with subsequent frozen-section histology, technetium- 99m sulphacolloid liver scan, liver function tests (serum albumin, total protein, bilirubin, alkaline phosphatase, aspartate transaminase, gamma-glutamyl transpeptidase and prothrombin time), laparoscopy, technetium-99m methylene diphosphonate bone scan, chest X-ray and X-ray of the lumbosacral spine and pelvis. Hot areas on the isotope bone scan were further investigated by radiology, and areas of increased uptake of the isotope were interpreted as deposits only if the X-ray films of the region showed no evidence of benign disease or trauma.

Both patients and control subjects were put on a gelatin-free diet for 2 days. The blood samples for OHPro studies were taken at the end of this period. None of the patients or controls admitted to the study had any concurrent disease known to affect the turnover of collagen and the excretion of OHPro in the urine. Blood was collected in EDTA for the estimation of plasma OHPro. The plasma was separated and stored at -20 "C until required.

The procedure of Le Roy et al. (1964) was modified for the estimation of plasma-bound OHPro. One millilitre of plasma and 4.0 ml of ethanol were mixed and the solution was left to stand at room temperature for 15 min. It was then centrifuged at 2127 g for 10 min. The protein residue was separated from the ethanol layer.

To the protein precipitate, 20 ml 12 M HC1 were added and the mixture was hydrolysed at 120 "C and 12 psi, for 18 h. The hydrolysate was evaporated to dryness and then dissolved in 1.5 ml of distilled water. One millilitre of the mixture was transferred to the cation-exchange chromatography column. The eluate solution was collected, neutralized, evaporated to dryness and then dissolved in 1.0 ml distilled water. Grant's method (1964) was used to measure OHPro. ~

* Department of Surgery, Ninewells Hospital and Medical School, Dundee, DDl 9SY Correspondence to: Professor A. Cuschieri, Department of Surgery, University of Dundee, Ninewells Hospital and Medical School, Dundee, DDl 9SY.

450 M. F. Nehlawi et al.

7

25

I I I 111 IV

50 75 100 125 150 175 Effluent volume (ml)

Fig. i. Hydroxyproline (OHPro) in protein fractions from plasma of healthy female volunteers.

I 0 1

25

I II 111 I V

I:: j i , I I I I .O

0 a

Effluent volume (rnl)

Fig. 2. Hydroxyproline (OHPro) in protein fractions from plasma of patients with early breast cancer (gro~ip A).

0- 25

I I I 111 1

IV

Fig. 3. Hydroxyproline (OHPro) in protein fractions from plasma of patients with advanccd breast cancer (group B).

PIasma proteins were fractionated using a Sephadex G200 gel filtration. The column height was 90cm and its diameter 1.5 cm. Four-rnillilitre plasma samples were run in a buffer containing 0.154 M NaC1 and 001 M Tris at pH 8.0. Sodium azide (0.02 per cent) was added to the solution to avoid bacterial growth. A flow rate of 4.0ml/h was adopted (bed volume, 135 ml). Fractions of 5.0 ml each were collected for about 36 h in an automated fraction collector (LKB). The proteins were measured by UV absorption at 280nm. Two millilitres of 12 M HCI were added to 4.0 ml of each protein fraction, hydrolysis was then performed and this was followed by cation-exchange chromatography. The OHPro content of each fraction was then measured.

TABLE I: TOTAL BOUND PLASMA HYDROXY- PROLINE

Group No. Bound OHPro (pmolll) Healthy 24 138.22~ 1.18 Benign breast disease 14 134.4+3.15

144.4+1.5* Breast cancer group A 15 Breast cancer group B 17 159-9&2.16*

(8ks . e . mean.) * PtO 05 (rank sum test).

TABLE 11: PERCENTAGE PROTEIN 1N GEL FILTRATION FRACTIONS

Grouv No. Fraction I Fraction 11

Healthy females 14 23.9k0.96 34.5 rt I .60 Benign breast disease 10 27.5&0.81** 38.7rt2.58 Breast cancer

Group A 15 29.5&1.18** 31.9f1.53 Group B 17 26.5k1.26 35 5f1.07

Fraction 111 Fraction IV Healthy females 14 31.8&1.42 9.2k0.80 Benign breast disease I0 24.8f2.16* 8.1 f0.35 Breast cancer

Group A I5 28.3&1.76* 9.0&1.01 17 28.9&1.79* 8.2h0.96 Group B

__.__.______-_I...---- ~

(X&s.e . mean.) * P < O 05; * * f ’ < O . O I (rank sum test).

Statistical analysis As the data obtained were not of a normal distribution, all statistical comparisons were carried out by non-parametric analysis using the rank slim test.

Results The total bound OHPro (peptide and protein) in the plasma (Table 1) was significantly elevated in patients with breast cancer compared with healthy controls and patients with benign breast disease. I n general, the high levels were obtained in patients with advanced disease (group R). However, the wide scatter of these results did not allow any eff’ective discrimination between breast cancer and the other groups.

‘The protein content of the various gel filtration fractions is shown in Table I I . Minor differences from control values were found in both patients with benign breast disease and patients with breast cancer but there was considerable overlap between the various groups. Small but significant elevations of the protein content of fraction I were observed in both patients with benign breast disease and patients with early breast cancer (group A). A reduced protein content of fraction 111 was found in patients with breast cancer but a greater reduction was observed in the protein content of the same fraction in patients with benign breast disease.

Typical examples of the plasma protein separation patterns and the OHPro content of the various fractions in healthy females, group A and group B breast cancer patients are shown in Fi-qs. 1, 2 and 3. The peaks of OHPro and the protein fractions were coincident. However, an additional OHPro peak was found located in between gel filtration fractions I11 and IV. There was no qualitative difference in either the protein separation or the distribution of OHPro between the various groups. The OHPro content of the various gel filtration fractions is shown in Table III.

Plasma-bound hydroxyproline in breast disease 451

Table 111: HYDROXYPROLINE (pnol/l) IN GEL FILTRATION FRACTIONS Separate OHPro

peak between Grouo No. Fraction I Fraction 11 Fraction 111 fractions 111 and IV Fraction 1V

Healthy females 14 51.1f3.29 36.8*2.20 27.91 1.66 5.4 f 1.02 15.012.43 Benign breast disease I0 49.715.12 30.3 & 1.84 21.2f 1.80 10.6 h2.40 17.3rt4.63 Breast cancer

Group A 15 40.4&2.80* 35.1rt2.34 29.4Ik3.24 14.lf2.40** 26.9Ik2.86: Group B 17 40.9+2.62* 37.1-+ 1.98 33.61 1.55 186*1.60** 29.7 i 2-19**

R fs.e. mean). * PtO.05; ** Pt0.01 (rank sum test).

Table IV: HYDROXYPROLINE SPECIFIC ACTIVITY IN GEL FILTRATION FRACTIONS

Group Healthy females Benign breast disease Breast cancer

Group A Group B

Healthy females Benign breast disease Breast cancer

Group A Grouo B

No. 14 10

15 17

14 10

15 17

Fraction I 2.85 k0.15 2.60&0.16

2.07 % 0.08 * * 2.2710.09**

Fraction 111

1.31 &0.08 1. I7rt0.06

Fraction I 1 1.61It0.13 1.1 7+0.07*

1.65 f 0.09 1.52 & 0.08

Fraction 1V 2.02 i 0.25 2. I2 & 0.65

4.04&0.52** 5.60&0.62**

(X+s.e. mean.) * P<0.025; ** P<0.005 (rank sum test).

A marked elevation of the OHPro content of fraction IV (peptide fraction) was observed in patients with breast cancer. The level of this peptide-bound plasma OHPro clearly separated the breast cancer patients from both patients with benign disease and healthy female controls. In addition, the OHPro peak between gel filtration fractions 111 and I V was significantly elevated only in patients with breast cancer. The OHPro content of both fraction IV and the separate OHPro peak in between fractions 111 and I V appeared to relate with degree of advancement of the disease. A significant reduction of the OHPro content of fraction I was found in patients with cancer of the breast.

As there were quantitative variations of protein in the various fractions, the specific activity of OHPro in each gel filtration fraction was calculated and expressed as mmol OHPro/g protein. These results are shown in Table ZV and demonstrate a significant elevation of the specific activity of OHPro in fraction I V and a decreased specific activity in fraction I in patients with cancer of the breast.

Discussion In addition to the free imino acid, OHPro is found in the human plasma in the peptide-bound form and as a component of certain proteins known as hypro- proteins. The high molecular weight hyproprotein which accounts for 55 per cent of the total is eluted from Sephadex columns with the early protein fractions containing the a2-macroglobulins and a- and P-lipoproteins. A smaller later peak of hyproprotein has been recovered from the albumin fraction (Le Roy et al., 1964).

Although the total bound OHPro in the plasma was found to be elevated in patients with breast cancer, there was considerable overlap between the groups

studied, including the healthy female controls. There were no qualitative differences from the normal pattern of distribution of OHPro amongst the plasma protein fractions in patients with either benign breast disease or breast cancer.

One of the significant quantitative changes from the normal observed in the breast cancer patients was a reduction of the high molecular weight hyproprotein in fraction I, despite the elevation of the total protein content of this fraction from the plasma of patients with breast cancer. The interpretation of this finding is difficult as both the exact nature and the pathological significance of hyproproteins remain to be determined. The suggestion that they represent collagen released from cells (Le Roy et al., 1964, 1965; Oh et al., 1968) is based on indirect evidence and the chemical identity between hyproprotein(s) and collagen has not been established. Even if they are associated with the metabolism of collagen, hyproproteins constitute a different pool from the peptide-bound OHPro as Le Roy et al. (1965) reported a total lack of corre- lation between hyproprotein levels and urinary peptide OHPro. Elevated levels of plasma hypro- protein have been found in the acute phase of a variety of disease states such as inflammatory and febrile illnesses, renal disease, rheumatoid arthritis etc. I t could be argued that plasma hyproproteins are acute phase reactants akin to C-reactive protein, haptoglobin and pre-albumin. There is some evidence based on animal experiments with C14-proline and a positive correlation between hyproprotein content and IgM levels in humans that the high molecular weight hyproprotein contains the immunoglobulin IgM (Tomaszewski and Kowalewski, 1975).

The plasma peptide-bound OHPro has received very little attention. It accounts for 6-10 per cent of the total OHPro in the plasma of healthy adults. Isotopic studies have shown that peptide-bound OHPro in the urine is derived from the turnover and degradation of both soluble and insoluble collagen (Prockop, 1963). In the present study, a significant elevation of the OHPro content of fraction I V was found only in patients with breast cancer. This gel filtration fraction reflects the polypeptide Component of the plasma and its OHPro content is, therefore, in the peptide-bound form. Patients with extensive locoregional disease and/or evidence of spread tended to have higher levels than patients with early breast cancer although there was overlap between these two subgroups. As it is extremely likely that some of the patients assessed as having early disease will have occult spread despite attempts at accurate staging, a valid assessment on the value of plasma peptide- bound OHPro as a prognostic index and as a monitor of disease activity in cancer of the breast can only be

452

based on the subsequent clinical outcome of these patients. We have as yet no definite information on any relation between plasma peptide-bound OHPro and survival in breast cancer as the period of follow-up for the patients included in this study has ranged from 6 months to 2 years.

A distinct peak of OHPro was observed in between gel filtration fractions I11 and IV. This is likely to represent a protein sub-unit rich in OHPro. A significant increase i n this OHPro peak was found in cancer of the breast, especially in patients with advanced disease. Further studies including purifi- cation, degradation and high pressure liquid chronia- tography are being performed in our laboratory to determine the exact nature of this moiety.

Whilst some of the changes reported in this study may simply reflect an increased turnover of collagen by metastatic tumour, their protein nature cannot be fully explained by this simple hypothesis. It seems logical to assume that the increased collagen turnover is not a local tissue response, but a component of a systemic reaction to an advancing neoplastic process characterized by complex changes in the plasma proteins. There is some support for this concept from recent studies in lymphoma patients where an increased excretion of urinary peptide OHPro was found exclusively in patients with systemic manifes- tations and related closely with pyrexia, elevated ESR and weight loss (Nehlawi et al., 1979).

Acknowledgement This work was supported by a grant from the Cancer Research Campaign.

References

M. F. Nehlawi et al.

BENOIT F. L., THElL G. 8. and WATTEN 11. H. (1963) Hydroxy- proline excretion i n endocrine disease. M&~bol i .~~r 12, 1072-1082.

BRUNISH R . and SBRENSEN B. (1965) Urinary excretion of acid mucopolysaccharides and hydroxyproline in psoriasis. Derniarologico (Easel) 130, 165-172.

CUSCHIEKI A. (1973) Urinary hydroxyproline excretion in early and advanced breast cancer: a sequential study. Br. J. Surg. 60, 800-803.

GRANT R. A. (1964) Estimation of hydroxyproline by the auto analyser. J . Clin. Pathul. 17, 685-686.

LE ROY E. c., KAPLAN A , , UDENFRIEND s. et al. (1964) A hydroxy- proline-containing, collagen-like protein in plasma and a procedure for its assay. J . Biol. Chenr. 239,3350-3356.

LE noY E. c. and SIOERDSMA A. (1965) Clinical significance of a hydroxyproline-containing protein in human plasma. J . Clin. Invest. 44, 914919.

N I ~ H L A W I M. F., SHAW D., MiTcHELL P. E. G. et al. (1979) Urinary hydroxyproline excretion in patients with Hodgkin’s disease and non-Hodgkin’s lymphoma. Clici. Oncol. ( In the press.)

OH s. I . , N E I F B. J . and BLOCK W. u. (1968) Soluble collagen in human serum. C‘lin. Chim. Actu 21, 1-8.

PLATT W. D . , DOOLITTLE L. H. and HARTSHORN J. W . S. (1964) Urinary hydroxyproline excretion i n metastatic cancer. N . Engl. J . Med. 271, 287-290.

I’OWLES T. J . , LEESE c. L. and BONDY 1’. K . (1975) Hydroxy- proline excretion in patients with breast cancer and response to treatment. Er. Mecl. J . 2, 164-166.

PROCKOP D. I. (1963) Isotopic studies on collagen degradation and the urine excretion of hydroxyproline. J . Clin. Incesr. 43, 453-460.

PROCKOP D. J., K E ~ S E R H. R. and S J O E I ~ D S M A A. (1962) Gastro- intestinal absorption and renal excretion of hydroxy- proline peptides. Lancet 2, 527-528.

nosticon test in breast cancer. Clin. Oncul. 1 , 33-43. sMiLEy J . D. and ZIFF M. (1964) Urinary hydroxyproline excretion

and growth. Physiol. Rev. 44, 30-44. TOMASZEWSKI J. and KOWALEWSKI J . (1975) High-molecular-

weight hydroxyproline protein in relation to immuno- globulins M (IgM) in human blood serum. Clin. Chin]. Acfn 60, 185-189.

zin: M., KIBRICK A , , DRESNER E. et al. (1956) Excretion of hydroxyproline in patients with rheumatic and non- rheumatic diseases. J . Clin. Inccst . 35, 579-587.

ROBERTS J . G . , WILLIAMS M. and HENK H.. M. (1975) The hypr0-

Paper accepted 30 November 1978.